Methods for use of TSLP and agonists and antagonists thereof

ABSTRACT

Methods are disclosed herein for specifically inducing proliferation of CD4 +  T cells. The methods are of use in treating immunodeficiencies, such as an immunodeficiency produced by infection with an immunodeficiency virus, such as infection with a human immunodeficiency virus (HIV). The methods include contacting isolated mammalian CD4+ T cells with an effective amount of a thymic stromal derived lymphopoietin (TSLP) polypeptide or a therapeutically effective amount of nucleic acid encoding the TSLP polypeptide, thereby inducing proliferation of the T cells. Methods are also disclosed for treating an IgE mediated disorder, such as asthma. The methods include administering to a subject a therapeutically effective amount of a TSLP antagonist. Transgenic mice are also disclosed herein. The somatic and germ cells of these mice include a disrupted thymic stromal lymphopoietin receptor (TSLP) gene, the disruption being sufficient to inhibit the interaction of TSLP with its receptor, and a disrupted γ c  gene, the disruption being sufficient to reduce signaling through the γ c . The mice exhibit diminished thymic cellularity. Methods of using these mice for drug screening are also disclosed.

PRIORITY CLAIM

This claims the benefit of U.S. Provisional Application No. 60/555,898,filed Mar. 23, 2004, which is incorporated by reference in its entirety.

FIELD

This disclosure relates to the field of immunology, specifically to theuse of thymic stromal lymphopoietin (TSLP), or TSLP agonists, to induceT cell proliferation and to treat immunodeficiency disorders. Thisdisclosure also relates to the use of TSLP antagonists to treat anIgE-mediated disorder, such as asthma or rhino-conjunctivitis.

BACKGROUND

In recent years, a novel murine growth factor, designated thymic stromallymphopoietin (TSLP), has been isolated. TSLP has been demonstrated tohave a role in B cell development and maturation. The cytokine activityof murine TSLP is very similar to that of IL-7, which is required duringproliferation and survival of pre-B cells (Friend et al., Exp. Hematol.,22:321-328, 1994). In addition, mature B lymphocytes fail to develop inthe absence of either IL-7 or murine TSLP. It has further been shownthat murine TSLP can replace IL-7 in sustaining B cell proliferativeresponses (Ray et al., Eur. J. Immunol. 26:10-16, 1996). Studies withIL-7 receptor (IL-7R) knockout mice indicate that IL-7, TSLP, or both,play a role in controlling the rearrangement of the T cellreceptor-gamma (TCR.gamma) locus (Candeias et al., Immunology Letters57: 9-14, 1997). Human TSLP has been cloned and sequenced (see U.S. Pat.No. 6,555,520). There is a need to determine the role in of TSLP human Tcell development and maturation.

The primary immunologic abnormality resulting from infection by HIV isthe progressive depletion and functional impairment of T lymphocytesexpressing the CD4 cell surface glycoprotein (Lane et al., Ann. Rev.Immunol. 3:477, 1985). CD4 is a non-polymorphic glycoprotein withhomology to the immunoglobulin gene superfamily (Maddon et al., Cell42:93, 1985). Together with the CD8 surface antigen, CD4 defines twodistinct subsets of mature peripheral T cells (Reinherz et al., Cell19:821, 1980), which are distinguished by their ability to interact withnominal antigen targets in the context of Class I and Class II majorhistocompatibility complex (MHC) antigens, respectively (see Swain,Proc. Natl. Acad. Sci. 78:7101, 1981). For the most part, CD4 T cellsdisplay the helper/inducer T cell phenotype (Reinherz, supra), althoughCD4 T cells characterized as cytotoxic/suppressor T cells have also beenidentified (Thomas et al., J. Exp. Med. 154:459, 1981; Meuer et al.,Proc. Natl. Acad. Sci. USA 79:4395, 1982). The loss of CD4helper/inducer T cell function probably underlies the profound defectsin cellular and humoral immunity leading to the opportunistic infectionsand malignancies characteristic of the acquired immunodeficiencysyndrome (AIDS) (Lane et al., Ann. Rev. Immunol. 3:477, 1985). Studiesof HIV-I infection of fractionated CD4 and CD8 T cells from normaldonors and AIDS patients have revealed that depletion of CD4 T cellsresults from the ability of HIV-I to selectively infect, replicate in,and ultimately destroy this T lymphocyte subset (Klatzmann et al.,Science 225:59, 1984).

The widespread use of highly active antiretroviral therapy (HAART) hasdramatically improved the clinical course for many individuals infectedwith HIV (Berrey et al., J Infect Dis 183(10):1466, 2001). However,toxicities associated with long term HAART have put a high priority onthe design and development of less toxic therapies. Among the “nextgeneration” of anti-viral inhibitors is T-20 (Wild et al., Proc NatlAcad Sci USA 91(26):12676, 1994; Wild et al. Proc Natl Acad Sci USA89(21):10537, 1992). However, there remains an acute need for additionaltherapeutic agents that can be used alone or in combination with HAARTto increase CD4 activity and treat HIV-infected individuals.

SUMMARY

Methods are disclosed herein for specifically inducing proliferation ofCD4⁺ T cells. These methods are of use in increasing the absolute numberof CD4⁺ T cells, and in increasing the CD4/CD8 ratio. The methods are ofuse in treating immunodeficiencies, such as an immunodeficiency producedby infection with an immunodeficiency virus, such as infection with ahuman immunodeficiency virus (HIV). The methods include contactingisolated mammalian CD4+ T cells with an effective amount of a thymicstromal derived lymphopoietin (TSLP) polypeptide or a therapeuticallyeffective amount of nucleic acid encoding the TSLP polypeptide, therebyinducing proliferation of the T cells.

Methods are also disclosed for the treatment of an inflammatory disordersuch as asthma, allergic rhinitis, allergic dermatitis, and allergicconjunctivitis. In one embodiment, the inflammatory disorder is anIgE-mediated disorder, such as a pulmonary IgE mediated disorder. Forexample, the disorder can be asthma. The methods include administeringto a subject with an IgE-mediated disorder a therapeutically effectiveamount of an antagonist of TSLP, thereby ameliorating a sign or asymptom of the disorder.

Transgenic mice are also disclosed herein. The somatic and germ cells ofthese mice include a disrupted thymic stromal lymphopoietin receptor(TSLP) gene, the disruption being sufficient to inhibit the interactionof TSLP with its receptor, and a disrupted γ_(c) gene, the disruptionbeing sufficient to reduce signaling through the γ_(c). The mice exhibitdiminished thymic cellularity. Methods of using these mice for drugscreening are also disclosed.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1G are a graphs and digital images of results obtained fromTSLPR KO mice. FIG. 1A is a schematic diagram of the TSLPR targetingstrategy. The 6 kb BglII to Nhe 15′ and 3 kb Pvu II 3′ flanking regionsof the TSLPR gene were cloned 5′ and 3′ to the Neo gene. The targetingvector was linearized, electroporated into ES cells, and transfectedclones were screened using 5′ and 3′ probes (filled squares). FIG. 1B isa digital image of a Southern hybridization of the ES clones. Therestriction enzymes and fragment sizes are indicated (+/+ for wild-type,+/− for heterozygote). FIG. 1C is a digital image of the genotyping ofthe mice, which was performed as described in the Material and Methods(left panel). RT-PCR was performed using TSLPR internal primers todetect the TSLPR mRNA transcript thymus (right panel). FIG. 1D is a bargraph showing that TSLP significantly increased anti-CD3ε-inducedproliferation of WT splenocytes (p<0.001) but not of TSLPR KOsplenocytes. Results are expressed as mean fold induction±SEM (n=14).FIGS. 1E, 1F and 1G are plots of flow cytometric analysis showing nodifference in the CD4/CD8 profile of the total thymus (FIG. 1E, upperpanel), the CD25/CD44 profile of DN thymocytes (FIG. 1E, lower panel),or in spleen (FIG. 1F) and bone marrow (BM) (FIG. 1G) of wild-type andTSLPR deficient mice. Antibodies for the CD4⁺ and CD8⁺ surface markerswere used to evaluate the T cell populations in the thymus (FIG. 1E) andspleen (FIG. 1F, upper panel) while antibodies for B220⁺ and surfaceIgM⁺ were used to evaluate B cells populations in the spleen (FIG. 1F,lower panel) and BM (FIG. 1G).

FIGS. 2A-2C are a graph, plot and a table showing that TSLP can expandlymphocyte populations in WT mice. FIG. 2A is a graph of the absolutenumber of thymocytes, splenocytes and BM cells in WT mice injected dailywith PBS, IL-7, or TSLP for 1 and 3 weeks. One week of treatment withTSLP and IL-7 increased thymic cellularity (means±SEM of 204±46×10⁶ and195±22×10⁶ [p=0.01 and p=0.001, respectively] versus 139±13×10⁶ for PBStreated mice). One week of treatment with TSLP and IL-7 also increasedsplenic cellularity (means±SEM of 126±23×10⁶ and 118±13×10⁶ [p=0.004 andp=0.002, respectively] versus 80±12×10⁶ for the PBS treated mice). Nosignificant difference was observed in the BM. After 3 weeks oftreatment with TSLP or IL-7, the changes in cellularity of thymus,spleen, and BM were not significant. FIG. 2B is a plot of the resultsfrom flow cytometric analysis of the BM 1 and 3 weeks after injection ofWT mice with PBS, IL-7, or TSLP. FIG. 2C is a table listing thepercentages of populations shown in FIG. 2B.

FIGS. 3A-3D are a set of graphs and plots showing that TSLP is criticalfor optimal lymphopoiesis in the presence and absence of IL-7. WT andTSLPR KO mice were sub-lethally irradiated and injected three times aweek for four weeks with 1 mg of control or M25 anti-IL-7 mAbs. FIG. 3Ais a graph showing that TSLPR KO treated with control or anti-IL-7 mAbsdisplayed lower thymic cellularity (p<0.001 for both mAbs) and spleniccellularity (p=0.03 for anti-IL-7 and p=0.02 for control mAb) whencompared to WT littermates. FIG. 3B is a set of graphs of the absolutenumbers of thymic subpopulations, except for DN cells, were decreased inTSLPR KO mice as compared to WT mice (p<0.05). FIG. 3C is a graph of theabsolute numbers of CD4⁺ and CD8⁺ T cells and B cells in spleens ofirradiated animals. Mice lacking TSLPR had fewer lymphocytes than WTmice, when experiments were performed with either control or anti-IL-7mAbs (p<0.05). FIG. 3D is a set of plots showing B220 versus CD19 flowcytometric analysis of spleen and bone marrow from WT (upper panel) andTSLPR KO (lower panel) mice.

FIGS. 4A-4E are graphs, plots and digital images showing a comparison ofthe lymphoid development in γ_(c) KO mice versus γ_(c)/TSLPR DKO mice.FIG. 4A is a graph showing γ_(c)/TSLPR double KO (DKO) mice had lowerthymic, spleen and BM cellularities than did γ_(c) KO mice (thymus:mean±SEM of 12.3±6.7×10⁶ for γ_(c) KO mice versus 5.7±4.5×10⁶ forγ_(c)/TSLPR DKO mice [p=0.009]; spleen: mean±SEM of 43±17.7×10⁶ forγ_(c) KO mice versus 26.8±13.7×10⁶ for γ_(c)/TSLPR DKO mice [p=0.02];bone marrow: mean±SEM of 23.4±15.5×10⁶ for γ_(c) KO mice versus14.3±7.6×10⁶ for γ_(c)/TSLPR DKO mice [p=0.047]). All mice wereage-matched and no sex-related differences were noted. FIG. 4B is a plotof the results obtained from flow cytometric analysis of bone marrow(B220 versus CD43, upper panel) and peritoneal cavity lymphocytes (B220versus CD5, lower panel). FIG. 4C is a graph showing similar levels ofIgM in the serum of γ_(c) KO and γ_(c)/TSLPR double KO mice. FIG. 4D isa graph showing injection of 0.5 μg/day of TSLP (open rectangles)enhances lymphoid cellularity in γ_(c) KO mice. Treatment for 1 and 3weeks showed an enhanced cellularity in thymus (mean±SEM of 7.8±2.9×10⁶for PBS-injected mice versus 32±10×10⁶ for TSLP-injected [p<0.0001]after 1 week treatment and a mean±SEM of 3.5±2×10⁶ for PBS-injected miceversus 12.2±4×10⁶ for TSLP-injected [p=0.001] after 3 weeks) and spleen(mean±SEM of 8±4×10⁶ for PBS-injected mice versus 14±5.4×10⁶ forTSLP-injected [p=0.003] after 1 week treatment and a mean±SEM of15±4×10⁶ for PBS-injected mice versus 52±12×10⁶ for TSLP-injected[p<0.0001] after 3 weeks). No change was seen in the BM. All mice wereage-matched and no sex-related differences were noted. γ_(c)/TSLPR DKOmice did not respond to TSLP injections (filled triangles). FIG. 4E is aset of digital images showing thymic size in γ_(c) KO mice after 1 weektreatment with PBS or TSLP (upper panel) and a histological analysis ofthese tissues by H&E staining (lower panel).

FIGS. 5A-5E are a set of images showing TSLP can increase lymphoidsubpopulations in γ_(c) deficient mice. FIG. 5A is a set of plots of theresults from flow cytometric analysis of γ_(c) KO thymus, spleen, and BM1 and 3 weeks after injection of PBS or TSLP. FIG. 5B is a table showingB cell populations in BM from FIG. 1A, subpanels vii, viii, xv, and xvi.FIG. 5C is a graph showing that TSLP injections induced an increase inCD4⁺ T cells (mean±SEM of 2.36±1.48×10⁶ for control mice versus12.7±6.7×10⁶ for TSLP treated mice [p<0.0001] for a 5.4 fold increase),CD8⁺ T cells (mean±SEM of 0.63±0.43×10⁶ for control mice versus2.4±1.7×10⁶ for TSLP treated mice [p=0.01] for a 3.8 fold increase) aswell as B cells (mean±SEM of 4.6±1.2×10⁶ for control mice versus27±7×10⁶ for TSLP treated mice [p<0.0001] for a 5.9 fold increase) inthe spleen of γ_(c) KO mice, 3 weeks following injection. FIG. 5D is aset of plots of the results from flow cytometric analysis of splenicCD4⁺ T cells in γ_(c) mice treated with PBS or TSLP for 3 weeks. TSLPincreased the absolute numbers of CD44^(high) CD62L^(low) (mean±SEM of2.3±1.2×10⁶ for control γ_(c) mice versus 11.3±4.1×10⁶ for TSLP treatedmice [p=0.0004] for 4.9 fold increase), CD44^(high) CD62L^(high)(mean±SEM of 0.4±0.2×10⁶ for control γ_(c) mice versus 3.2±1.1×10⁶ forTSLP treated mice [p=0.0003] for an 8 fold increase) and CD44^(low)CD62L^(high) (mean±SEM of 0.08±0.06×10⁶ for control γ_(c) mice versus0.9±0.16×10⁶ for TSLP treated mice [p<0.0001] for an 11 fold increase).FIG. 5E is a set of plots of the results of experiments wherein γ_(c) KOmice were treated with PBS or TSLP for 1 week, injected with BrdU 10 and16 hours before sacrifice. BrdU incorporation was measured byintracellular staining using PE-labeled BrdU of the thymocytessubpopulations. The number indicates the percent of BrdU⁺ cells withinthe gated region.

FIGS. 6A-6C are a set of bar graphs and plots showing that TSLPpreferentially expands CD4⁺ T cells. For the results shown in FIG. 6Aand FIG. 6B, cells were treated with medium, IL-7 (100 ng/ml), or TSLP(100 ng/ml) and/or anti-CD3ε antibodies (2 μg/ml). FIG. 6A is a bargraph showing the in vitro proliferation of purified CD4⁺ and CD8⁺ SPthymocytes from WT mice. Results are expressed as mean±SEM for 4experiments. TSLP increased anti-CD3ε induced proliferation of CD4⁺ SPcells (p=0.02) but did not significantly affect CD8⁺ SP expansion toanti-CD3ε (p=0.07). IL-7 significantly enhanced anti-CD3ε inducedexpansion of both CD4⁺ and CD8⁺ SP thymocytes (p<0.0001 for both). FIG.6B is a bar graph showing in vitro proliferation of purified CD4⁺ andCD8⁺ splenocytes (prepared by positive selection) treated as describedabove. TSLP significantly increased anti-CD3ε-induced proliferation ofmature CD4⁺ T cells (p=0.0002) but not of CD8⁺ T cells (p=0.1). IL-7significantly enhanced anti-CD3ε induced expansion of both CD4⁺ and CD8⁺mature T cells (p=0.008 and p=0.0005 respectively). FIG. 6C is a set ofplots showing the results obtained when CD4⁺ thymocytes and splenic Tcells were labeled with CFSE and cultured for 1 week with anti-CD3ε withor without TSLP, and cells were analyzed by flow cytometry. As evaluatedby decreased CFSE staining, TSLP increased anti-CD3ε-inducedproliferation of CD4⁺ but not of CD8⁺ T cells. WT corresponds to the“open” curve, whereas TSLPR KO mice are shown in solid black.

FIGS. 7A-7C are a set of bar graphs and plots showing that TSLP promotessurvival of CD4⁺ T cells. FIG. 7A is a bar graph demonstrating the invitro survival of purified CD4⁺ and CD8⁺ SP thymocytes from WT mice. Thepercent viable cells (mean±SEM for 4 experiments) was determined after 1week by trypan blue exclusion. TSLP increased anti-CD3ε induced survivalof CD4⁺ cells (p=0.02) but did not significantly affect CD8⁺ survival(p=0.24). IL-7 significantly enhanced anti-CD3ε induced survival of bothCD4⁺ and CD8⁺ thymocytes (p=0.03 and p<0.0001, respectively). FIG. 7B isa bar graph showing the results obtained from an in vitro survival assayof purified CD4⁺ and CD8⁺ splenic T cells from WT mice. The percentageof viable cells was determined by trypan blue exclusion. Results areexpressed as mean±SEM for 5 experiments. TSLP significantly increasedanti-CD3ε induced survival of CD4⁺ T cells (p<0.0001) but not of CD8⁺ Tcells (p=0.21). IL-7 significantly enhanced anti-CD3ε induced survivalof both CD4⁺ and CD8⁺ T cells (p<0.001 and p<0.0001, respectively). FIG.7C is a set of plots showing purified CD4⁺ and CD8⁺ splenocytes culturedas indicated were stained with Annexin V and 7-AAD.

FIGS. 8A-8B are a graph and a set of plots showing that TSLP mediatesefficient expansion of CD4⁺ T cells. CD4⁺ T cells were isolated from WTor TSLPR KO mice and labeled with CFSE before being injected intoirradiated γ_(c) KO mice. FIG. 8A is a graph showing that after 1 week,TSLPR KO CD4⁺ T cells expanded less than CD4⁺ T cells from WT mice(p=0.008). CD8⁺ T cells from WT or TSLPR mice expanded to a similardegree. FIG. 8B is a set of plots showing that examination of the CFSEdilution on day 3 by flow cytometry revealed that WT CD4⁺ T cells wereexpanding more rapidly than TSLPR KO CD4⁺ T cells. No differences wereobserved for CD8⁺ T cells.

FIGS. 9A-9B are a set of plots showing the effect of TSLP onlymphopoiesis in WT mice. Flow cytometric analysis of thymus and spleen1 (FIG. 9A) and 3 weeks (FIG. 9B) after injection of WT mice with PBS,IL-7, or TSLP.

FIGS. 10A-10E are a set of graphs and plots showing TSLP promotes theproliferation of naive CD4⁺ T cells. For the results shown in the bardgraph shown in FIG. 10A, purified naïve (CD4⁺CD62L⁺CD44⁻), centralmemory (CD4⁺CD62L⁺CD44⁺), and effector memory (CD4⁺CD62L⁻CD44⁺) T cellsfrom WT Balb/c mice were activated for 48 hours with 2 μg/ml anti-CD3with or without 100 ng/ml TSLP and then pulsed with ³H thymidine for 16hours. TSLP significantly increased the proliferation of naïve CD4⁺ Tcells (p=0.01) (upper panel) but did not significantly affect memoryCD4⁺ T cells proliferation (middle and lower panels). Lowerconcentrations of anti-CD3 (0.2-0.5 μg/ml) had a similar preferentialeffect on naive CD4⁺ T cells (data not shown). For the results shown inthe plots shown in FIG. 10B, WT and TSLPR KO mice expressing the DO11.10transgene were injected with OVA and ALUM (i.p.) and analyzed the nextday. DO11.10/WT but not DO11.10/TSLPR KO mice expressed high levels ofCD69. For the results shown in the graphs of FIGS. 10C, 10D and 10E, WTand TSLPR KO mice (not expressing the DO11.10 transgene) were immunizedwith ovalbumin (OVA) (200 μg) and ALUM. Animals were sacrificed on day12 (FIG. 10C) or 60 (FIG. 10D) and splenocytes were cultured with OVA(0, 10, 50, and 200 μg/ml). Splenocytes from TSLPR KO mice displayedsignificantly lower proliferation in response to secondary encounterwith antigen at all concentrations tested (p<0.05 for all). For theresults graphed in FIG. 10E, CD4⁺ T cells purified from mice 60 daysafter immunization were incubated with APC (splenocytes that weredepleted of T and NK cells) in the presence of different doses of OVA.CD4⁺ T cells from TSLPR KO mice displayed significantly lowerproliferation in response to the OVA (200 μg/ml) in vitro (p<0.01) thanCD4⁺ T cells from WT mice. Shown are means±SEM for 5 experiments.

FIGS. 11A-11D are a set of images showing that TSLP activates murine DC.FIG. 11A is a graph of the results of in vitro antigen recall response.CD4⁺ T cells and APC from immunized WT and TSLPR KO mice were culturedwith 200 μg/ml of OVA for 48 hours before being pulsed with ³Hthymidine. Replacing WT-APC with TSLPR KO-derived APC reduced theantigen-driven proliferation of WT CD4⁺ T cells. However WT-APC did notrescue the defected expansion of TSLPR KO CD4⁺ T cells. Shown aremeans±SEM for 7 experiments. For the plots shown in FIG. 11B, splenicCD11c⁺ cells were sorted from the spleens of WT Balb/c animals andincubated overnight with 5 μg/ml of OVA³²³⁻³³⁹ peptide alone or withTSLP. TSLP treatment increased the surface levels of CD80, MHC class II,and CD86 as compared to peptide alone. For the bar graph shown in FIG.11C, sorted splenic DC were incubated with 5 μg/ml of OVA³²³⁻³³⁹ peptidealone or with TSLP before being washed, treated with mitomycin C, andcultured with purified CD4⁺ T cells from DO1.10 RAG2^(−/−) mice at a1:10 ratio. Antigen presentation of DC was significantly enhanced byTSLP treatment (means±SEM for 5 experiments). For the results graphed inFIG. 11D, CD4⁺ T cells and DC were purified from DO11.10/WT andDO11.10/TSLPR KO unmanipulated mice, and these were cultured together inthe indicated combinations at a ratio of 1:10 DC:T cell, with 5 μg/ml ofOVA³²³⁻³³⁹ peptide. Proliferation after 48 hours was examined bymeasuring ³H-thymidine incorporation. Replacing WT-APC with TSLPRKO-derived DC moderately but significantly reduced the antigen-drivenproliferation of DO11.10 Tg/WT CD4⁺ T cells (p=0.04). WT-DC provided nosignificant enhancement over the weak expansion of DO11.10/TSLPR KO CD4⁺T cells. Shown are means±SEM for 7 experiments. In this figure, *indicates statistical significance p<0.05.

FIGS. 12A-12F are a set of images showing that DC activated with TSLPnegatively regulates IFN-γ production by naïve CD4⁺ T cells. Cells werecultured before being treated with PI, and the intracellular levels ofIFN-γ and IL-4 were measured by intracellular staining. For the plotsshown in FIG. 12A, naïve and memory CD4⁺ T cells were isolated (>99%pure) from WT animals and treated with anti-CD3 with or without TSLP for4 days. The addition of TSLP had no effect on the levels of IFN-γ andIL-2 produced by CD4⁺ T cells. For the plots shown in FIG. 12B, purifiedCD4⁺ T cells were activated with anti-CD3/anti-CD28 under Th1 (IL-12 andanti-IL-4) or Th2 (IL-4 and anti-IFN-γ) polarizing conditions with orwithout TSLP. IL-2 was added on day 2, and cells were allowed to growfor 1 week. TSLP did not affect the IFN-γ or IL-4 production by thesepolarized cells. For the plots shown in FIG. 12C, sorted splenic CD11c⁺DC were incubated with 5 μg/ml of OVA³²³⁻³³⁹ peptide alone or with TSLPbefore being washed, treated with mitomycin C, and cultured withpurified CD4⁺ T cells from DO11.10 RAG2^(−/−) mice at a 1:10 ratio fromnon-immunized animals. TSLP-treated DC reduced the levels of IFN-γproduction by KJ1-26⁺CD4⁺ T cells, whereas the levels of IL-4 were notaffected.

FIG. 12D is a bar graph showing that no significant difference wasobserved in the levels of IL-12 (p35) mRNA examined by RTPCR in CD11c⁺DC that were incubated overnight with 5 μg/ml of OVA³²³⁻³³⁹ peptidealone or with TSLP. For the plots shown in FIG. 12E, total splenocytesfrom DO11.10WT and DO11.10/TSLPRKO mice were cultured for 4 days with 5μg/ml of OVA³²³⁻³³⁹ peptide before being activated with PI for 5 h.KJ1-26⁺ TSLPR KO T cells produced more IFN-γ than DO11.10/WT cells. Forthe bar graph shown in FIG. 12F, RNA was extracted from DO11.10/WT andDO11.10/TSLPRKO total splenocytes that were cultured for 4 days with 5μg/ml of OVA³²³⁻³³⁹ peptide. RTPCR revealed significantly lower levelsof IL-4 transcripts in the spleens of TSLPR KO mice (p<0.05).

FIGS. 13A-D are a set of digital images showing that TSLPR KO mice failto mount an inflammatory response. PAS-stained lung tissue sections (40×magnification) of Balb/c WT and TSLPR KO mice that were sensitized(i.p.) and challenged (i.t. and i.n.) with OVA or PBS (i.p.). Upperpanels display no obvious differences in the lung morphology between WTand TSLPR KO animals in the resting state. WT mice receiving OVAdisplayed perivascular, peribronchiolar cuffing, and goblet cellhyperplasia (lower left panel), whereas TSLPR KO mice treated with OVAshowed no obvious inflammation (lower right panel).

FIGS. 14A-14B are a set of bar graphs showing the absence of TSLPRinhibits allergic/inflammatory immunoglobulin and cytokine profiles inthe lungs. Balb/c WT and TSLPR KO mice were sensitized (i.p.) andchallenged (i.t. and i.n.) with OVA or PBS (i.p.). FIG. 14A is a set ofbar graphs showing serum levels of OVA-specific immunoglobulin in PBSand OVA treated mice. OVA-TSLPR KO displayed significantly less IgE andmore IgG2a than WT treated littermates. FIG. 14B is a set of bar graphsshowing cytokine levels in the lungs as determined by RT-PCR. Theresults are expressed as means±SEM for 4 experiments; * indicatesstatistical significance p<0.05.

FIGS. 15A-15C are a set of digital images showing TSLPR KO mice succeedin mounting an inflammatory response in the lung after receiving WT CD4⁺T cells. Donor and recipient mice (F4 Balb/c) were sensitized with OVA(i.p.). Recipient mice were challenged with OVA (i.t.) 4 hours beforereceiving 8×10⁶ of total CD4⁺ T cells. CD4⁺ T cells from WT donor weretransferred to WT (positive control) and TSLPR KO (test subject) micewhile CD4⁺ T cells from TSLPR KO mice were given to TSLPR KO mice to actas negative control. All mice were treated the next day with OVA (i.n.)and sacrificed 24 hours later. Shown are the PAS staining of lung tissuesections at 10× and 40× magnification. The WT positive control displayedperivascular, peribronchiolar cuffing, and goblet cell hyperplasia (FIG.15A) while TSLPR KO mice showed no signs of lung inflammation (FIG.15B). However, TSLPR KO mice that were supplemented with CD4⁺ T cells(FIG. 15C) exhibited inflammatory cells infiltration combined withperibronchiolar cuffing, and goblet cell hyperplasia in the large andmedium airways.

FIG. 16 is a set of bar graphs showing inflammatory cytokine levels inTSLPR KO mice supplemented with WT CD4⁺ T cells. CD4⁺ T cells wereextracted from immunized donors and transferred to host mice 4 hoursafter OVA challenge (i.t). CD4⁺ T cells from WT donors were transferredto WT (positive control) and TSLPR KO (test subject) mice while CD4⁺ Tcells from TSLPR KO mice were given to TSLPR KO mice to act as negativecontrol. All mice were treated the next day with OVA (i.n.) andsacrificed 24 hours later. RNA was isolated from lung tissues andcytokine levels were determined by RTPCR. * indicates statisticalsignificance p<0.05 between TSLPR KO mice that received WT CD4⁺ T cellsand those that did not.

FIG. 17 is Table 3.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. In the accompanying sequence listing:

SEQ ID NOs: 1-5 are amino acid sequences of TSLP polypeptides.

SEQ ID NOs: 6-10 are the nucleic acid sequences of primers or probes.

SEQ ID NO: 11 is an exemplary sequence of a polynucleotide encodingTSLP.

DETAILED DESCRIPTION

I. Abbreviations

-   -   γ_(C): common cytokine receptor gamma (γ) chain    -   Ab: antibody    -   APC: antigen presenting cell    -   CDR: complementarity-determining regions    -   CFSE: carboxy fluorescein diacetate succinimide ester    -   DC: dendritic cell    -   DN: double negative    -   DP: double positive (for example CD4⁺CD8⁺)    -   ES: embryonic stem cells    -   FACS: fluorescence activated cell sorting or scanning    -   HIV: human immunodeficiency virus    -   Hr or h: hour    -   IFN: interferon    -   Ig: immunoglobulin    -   IL: interleukin    -   i.p.: intraperitoneal    -   i.v.: intravenous    -   JAK: Janus Activated Kinase    -   kb: kilobases    -   KO: knock-out    -   min: minutes    -   NK: natural killer cell    -   OVA: ovalbumin    -   PBS: phosphate buffered saline    -   PCR: polymerase chain reaction    -   S.D.: standard deviation    -   sec: seconds    -   SCID: severe combined immunodeficiency disease    -   SP: single positive (for example, either CD4⁺ or CD8⁺)    -   STAT: Signal Transducer and Activator of Transcription    -   TCR: T cell receptor    -   TG or Tg: transgenic    -   TSLP: thymic stromal lymphopoietin    -   TSLPR: thymic stromal lymphopoietin receptor    -   μg: microgram    -   vs: versus    -   WT: wild-type        II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Agent: Any polypeptide, compound, small molecule, organic compound,salt, polynucleotide, or other molecule of interest.

Agonist/Antagonist: An agent that has affinity for and stimulatesphysiologic activity at cell receptors normally stimulated by naturallyoccurring substances, thus triggering a biochemical response. A TSLPreceptor agonist has affinity for the TSLP receptor and stimulates anactivity induced by the binding of TSLP with its receptor. For example,a TSLP/TSLP receptor agonist is a molecule that binds to the TSLPreceptor and induces intracellular signaling. In contrast, an“antagonist” is an agent that inhibits activity of a cell receptornormally stimulated by a naturally occurring substance. Accordingly, aTSLP/TSLP receptor antagonist binds to TSLP or to the TSLP receptor andinhibits binding of TSLP to the TSLP receptor and/or inhibits anactivity normally induced by binding of TSLP with its receptor. Forexample, a TSLP/TSLP receptor antagonist can bind to TSLP or to the TSLPreceptor and diminish or prevent binding, for example, by blockingbinding, of TSLP to the TSLP receptor. Alternatively, a TSLP/TSLPreceptor antagonist can bind to the TSLP receptor and diminish orprevent downstream signaling that would normally be induced by thebinding of TSLP with its receptor. Agonists and antagonists can includea variety of classes of molecules including polypeptides, such asligand-like polypeptides, antibodies, and fragments or subsequencesthereof. Agonists and antagonists can also include fusion polypeptides,antibodies, peptides (such as peptides of less than about 20 amino acidsin length), and small molecules. Exemplary antagonists include:neutralizing antibodies specific for TSLP and the TSLP receptor, solubleTSLP receptor molecules, and TSLP receptor fusion proteins, such asTSLPR-immunoglobulin Fc molecules or polypeptides that encode componentsof more than one receptor chain, that thereby mimic a physiologicalreceptor heterodimer or higher order oligomer. If the receptor is aincludes more than one polypeptide chain, a single chain fusion can beutilized.

Animal: A living multicellular vertebrate organism, a category whichincludes, for example, mammals and birds. A “mammal” includes both humanand non-human mammals. Similarly, the term “subject” includes both humanand veterinary subjects.

Antibody: A polypeptide ligand comprising at least a light chain orheavy chain immunoglobulin variable region which specifically recognizesand binds an epitope (e.g., as an antigen, such as TSLP or a fragmentthereof, or a TSLP receptor of a fragment thereof). This includes intactimmunoglobulins and the variants and portions of them well known in theart, such as Fab′ fragments, F(ab)′₂ fragments, single chain Fv proteins(“scFv”), and disulfide stabilized Fv proteins (“dsFv”). A scFv proteinis a fusion protein in which a light chain variable region of animmunoglobulin and a heavy chain variable region of an immunoglobulinare bound by a linker, while in dsFvs, the chains have been mutated tointroduce a disulfide bond to stabilize the association of the chains.The term also includes genetically engineered forms such as chimericantibodies (e.g., humanized murine antibodies), heteroconjugateantibodies (e.g., bispecific antibodies). See also, Pierce Catalog andHandbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J.,Immunology, 3^(rd) Ed., W.H. Freeman & Co., New York, 1997.

Typically, an immunoglobulin has a heavy and a light chain. Each heavyand light chain contains a constant region and a variable region, (theregions are also known as “domains”). In combination, the heavy and thelight chain variable regions specifically bind the antigen. Light andheavy chain variable regions contain a “framework” region interrupted bythree hypervariable regions, also called “complementarity-determiningregions” or “CDRs”. The extent of the framework region and CDRs has beendefined (see, Kabat et al., Sequences of Proteins of ImmunologicalInterest, U.S. Department of Health and Human Services, 1991, which ishereby incorporated by reference). The Kabat database is now maintainedonline. The sequences of the framework regions of different light orheavy chains are relatively conserved within a species. The frameworkregion of an antibody, that is the combined framework regions of theconstituent light and heavy chains, serves to position and align theCDRs in three-dimensional space.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso typically identified by the chain in which the particular CDR islocated. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found.

References to “V_(H)” or “VH” refer to the variable region of animmunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab.References to “V_(L)” or “VL” refer to the variable region of animmunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.

A “monoclonal antibody” is an antibody produced by a single clone ofB-lymphocytes or by a cell into which the light and heavy chain genes ofa single antibody have been transfected. Monoclonal antibodies areproduced by methods known to those of skill in the art, for instance bymaking hybrid antibody-forming cells from a fusion of myeloma cells withimmune spleen cells. Monoclonal antibodies include humanized monoclonalantibodies.

A “humanized” immunoglobulin is an immunoglobulin including a humanframework region and one or more CDRs from a non-human (such as a mouse,rat, or synthetic) immunoglobulin. The non-human immunoglobulinproviding the CDRs is termed a “donor,” and the human immunoglobulinproviding the framework is termed an “acceptor.” In one embodiment, allthe CDRs are from the donor immunoglobulin in a humanizedimmunoglobulin. Constant regions need not be present, but if they are,they must be substantially identical to human immunoglobulin constantregions, i.e., at least about 85-90%, such as about 95% or moreidentical. Hence, all parts of a humanized immunoglobulin, exceptpossibly the CDRs, are substantially identical to corresponding parts ofnatural human immunoglobulin sequences. A “humanized antibody” is anantibody comprising a humanized light chain and a humanized heavy chainimmunoglobulin. A humanized antibody binds to the same antigen as thedonor antibody that provides the CDRs. The acceptor framework of ahumanized immunoglobulin or antibody may have a limited number ofsubstitutions by amino acids taken from the donor framework. Humanizedor other monoclonal antibodies can have additional conservative aminoacid substitutions which have substantially no effect on antigen bindingor other immunoglobulin functions. Humanized immunoglobulins can beconstructed by means of genetic engineering (e.g., see U.S. Pat. No.5,585,089).

Antigen: A compound, composition, or substance that can stimulate theproduction of antibodies or a T cell response in an animal, includingcompositions that are injected or absorbed into an animal. An antigenreacts with the products of specific humoral or cellular immunity,including those induced by heterologous immunogens. The term “antigen”includes all related antigenic epitopes. An “antigen presenting cell” isa cell that presents an antigen to the immune system. There are threegeneral classes of antigen presenting cells (APCs): macrophages,dendritic cells, and B cells, although neutrophils can also presentantigens. Processing and surface presentation of antigen by APCs can bethought of as a first step in the normal immune response. The antigencan be any antigen, including but not limited to an antigen of abacterial, virus, fungus, or any other infectious organism.

Anti-Inflammatory Agent: Any of various medications that decrease thesigns and symptoms (for example, pain, swelling, or shortness of breath)of inflammation. Corticosteroids are exemplary potent anti-inflammatorymedications. Nonsteroidal anti-inflammatory agents are also effectiveexemplary anti-inflammatory agents and do not have the side effects thatcan be associated with steroid medications.

Asthma: A clinical syndrome characterized by recurrent episodes ofairway obstruction that resolve spontaneously or as a result oftreatment. The resolution of the airway obstruction is a feature thatdistinguishes it from forms of chronic obstructive pulmonary disease.Asthma is also associated with hyperresponsiveness of the airways to avariety of inhaled stimuli; this condition is manifested as anexaggerated bronchoconstrictor response to stimuli that have little orno effect in normal subjects. Asthma is sometimes referred to asreactive airway disease.

Episodic airway narrowing constitutes an “asthma attack,” and resultsfrom obstruction of the airway lumen to airflow. Three distinctpathological processes account for the obstruction: (1) constriction ofairway smooth muscle, (2) thickening of airway epithelium, and (3) thepresence of liquids within the confines of the airway lumen. It has beenhypothesized that constriction of airway smooth muscle is due to thelocal release of bioactive mediators or neurotransmitters. During anasthma attack, patients experience shortness of breath accompanied bycough, wheezing and anxiety. Dyspnea may occur with exercise. In oneembodiment, asthma is diagnosed by the presence of symptoms (such asrecurrent cough, wheezing or dyspnea), and the presence of reversibleairflow limitation (such as diminished forced expiratory volume in onesecond (FEV1), or in peak expiratory flow rate either spontaneously orwith an inhaled short-acting beta2-agonist), or increased airwayresponsiveness to methacholine).

Bronchodilator: An antispasmodic or other agent that dilates a bronchusor bronchiole. Bronchodilators relax the smooth muscles of the airways,allowing the airway to dilate. Bronchodilator medicines do notcounteract inflammation.

CD4: Cluster of differentiation 4 polypeptide, a T cell surface proteinthat mediates interaction with the MHC Class II molecule. CD4 alsoserves as the primary receptor site for HIV on T cells during HIVinfection.

The known sequence of the CD4 precursor has a hydrophobic signalpeptide, an extracellular region of approximately 370 amino acids, ahighly hydrophobic stretch with significant identity to themembrane-spanning domain of the Class II MHC beta chain, and a highlycharged intracellular sequence of 40 resides (Maddon, Cell 42:93, 1985).

CD8: Cluster of differentiation 8 polypeptide, a T cell surface proteinthat mediates interaction with MHC Class I molecule. CD8 occurs eitheras a disulfide-linked homodimer or homomultimer of two 34 kDa subunits(CD8α) or as a heterodimer complexed with another protein named CD8β, ofwhich there are multiple forms arising by alternative splicing of itsmRNA.

T cell progenitors in the thymus initially do not express CD8 or CD4.These progenitors develop into mature T cells in several steps. Immaturethymocytes coexpress CD8 and CD4 (DP or CD4⁺CD8⁺ cells), and these cellsgive rise to mature T cells which are either CD4⁺CD8⁻ or CD4⁻ CD8⁺ (alsocalled SP cells). Those T cells that recognize self-MHC are selected tomature by a process known as positive selection in which Class I MHCgenerates an instructive signal that directs differentiation to a CD8lineage.

cDNA (complementary DNA): A piece of DNA lacking internal, non-codingsegments (introns) and transcriptional regulatory sequences. cDNA canalso contain untranslated regions (UTRs) that are responsible fortranslational control in the corresponding RNA molecule. cDNA issynthesized in the laboratory by reverse transcription from messengerRNA extracted from cells.

Chronic Bronchitis: A long-standing inflammation of the airways thatproduces a lot of mucus, causing wheezing and infections. It isconsidered chronic if a subject has coughing and mucus on a regularbasis for at least three months a year and for two years in a row.

Chronic Obstructive Pulmonary Disease (COPD): COPD refers mainly to twoclosely related respiratory disorders that cause gradual loss ofpulmonary function: chronic bronchitis and emphysema associated withairflow obstruction. A subject with COPD sometimes has both chronicbronchitis and emphysema, or may just have one of these diseases.Chronic bronchitis is a long-standing inflammation of the airways thatproduces a lot of mucus, causing wheezing and infections. It isconsidered chronic if a subject has coughing and mucus on a regularbasis for at least three months a year and for two years in a row.Emphysema is a disease that destroys the alveolae and/or bronchae.Simply put, the lungs lose elasticity. This causes the air sacs tobecome enlarged, thus making breathing difficult.

In the beginning stages of COPD, a subject may have only a mildshortness of breath and occasional coughing spells. Initial symptoms caninclude a general feeling of illness, increasing shortness of breath,coughing and wheezing. But, as the disease progresses, symptoms becomeincreasingly more severe.

The majority of COPD subjects have a history of smoking. In addition,untreated or under-treated asthma may lead to irreversible lung damage.These subjects may have symptoms somewhat similar to COPD.

Comprises: A term that means “including.” For example, “comprising A orB” means including A or B, or both A and B, unless clearly indicatedotherwise.

Conservative substitutions: Modifications of a polypeptide that involvethe substitution of one or more amino acids for amino acids havingsimilar biochemical properties that do not result in change or loss of abiological or biochemical function of the polypeptide. These“conservative substitutions” are likely to have minimal impact on theactivity of the resultant protein. Table 1 shows amino acids that may besubstituted for an original amino acid in a protein, and which areregarded as conservative substitutions. Original Residue ConservativeSubstitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn GluAsp His Asn; Gln Ile Leu, Val Leu Ile; Val Lys Arg; Gln; Glu Met Leu;Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu

In one embodiment, one or more conservative changes, or up to tenconservative changes, can be made in a polypeptide without changing abiochemical function of the polypeptide. More substantial changes in abiochemical function or other protein features may be obtained byselecting amino acid substitutions that are less conservative than thoselisted in Table 1. Such changes include, for example, changing residuesthat differ more significantly in their effect on maintainingpolypeptide backbone structure (e.g., sheet or helical conformation)near the substitution, charge or hydrophobicity of the molecule at thetarget site, or bulk of a specific side chain. The followingsubstitutions are generally expected to produce the greatest changes inprotein properties: (a) a hydrophilic residue (e.g., seryl or threonyl)is substituted for (or by) a hydrophobic residue (e.g., leucyl,isoleucyl, phenylalanyl, valyl or alanyl); (b) a cysteine or proline issubstituted for (or by) any other residue; (c) a residue having anelectropositive side chain (e.g., lysyl, arginyl, or histadyl) issubstituted for (or by) an electronegative residue (e.g., glutamyl oraspartyl); or (d) a residue having a bulky side chain (e.g.,phenylalanine) is substituted for (or by) one lacking a side chain(e.g., glycine).

Cytokine/Interleukin (IL): A generic name for a diverse group of solubleproteins and peptides which act as humoral regulators at nano- topicomolar concentrations and which, either under normal or pathologicalconditions, modulate the functional activities of individual cells andtissues. These proteins also mediate interactions between cells directlyand regulate processes taking place in the extracellular environment.Many growth factors and cytokines act as cellular survival factors bypreventing programmed cell death. Cytokines and interleukins includeboth naturally occurring peptides and variants that retain full orpartial biological activity. Although specific cytokines/interleukinsare described in the specification, they are not limited to thespecifically disclosed peptides.

Cystic Fibrosis: A disease that most commonly affects the lungs anddigestive systems, especially the pancreas. It causes the exocrineglands, which produce mucus and sweat, to produce abnormal secretions.Cystic fibrosis causes the cells in the lung tissue to produce anabnormal amount of thick, sticky mucus that clogs the airways of thelungs, resulting in pulmonary obstructions and life-threateningbacterial infections.

DNA: Deoxyribonucleic acid. DNA is a long chain polymer which comprisesthe genetic material of most living organisms (some viruses have genescomprising ribonucleic acid (RNA)). The repeating units in DNA polymersare four different nucleotides, each of which comprises one of the fourbases, adenine, guanine, cytosine and thymine bound to a deoxyribosesugar to which a phosphate group is attached. Triplets of nucleotides(referred to as codons) code for each amino acid in a polypeptide. Theterm codon is also used for the corresponding (and complementary)sequences of three nucleotides in the mRNA into which the DNA sequenceis transcribed.

Deletion: The removal of a sequence of DNA, the regions on either sidebeing joined together.

Encode: A polynucleotide is said to “encode” a polypeptide if, in itsnative state or when manipulated by methods well known to those skilledin the art, it can be transcribed and/or translated to produce the mRNAfor and/or the polypeptide or a fragment thereof. The anti-sense strandis the complement of such a nucleic acid, and the encoding sequence canbe deduced therefrom.

Expectorant: A drug or chemical substance that induces the ejection ofmucus, phlegm and other fluids from the lungs and air passages, forexample by coughing.

Expiratory Flow Rate: The rate at which air is expelled from the lungsduring exhalation. A subject's maximum expiratory flow is measured by asimple pulmonary test; in performing the test, a subject first takes asdeep a breath as possible, then exhales as rapidly and as completely aspossible into a machine known as a spirometer, which measures the rateof exhalation. Forced expiratory flow 25-75 (FEF 25-75) is a measurementof the forced expiratory flow determined over the midportion of a forcedexhalation. An increase in the forced expiratory flow (FEF) or FEF 25-75reflects a decrease in bronchoconstriction and an improvement inpulmonary function.

Forced Expiratory Volume (FEV): The forced expiratory volume is thevolume of air resulting from the forced expiratory flow test in which asubject first inspires maximally to the total lung capacity, thenexhales as rapidly and as completely as possible. The forced expiredvolume in one second (FEV1) represents the maximum expiratory air volumea subject can produce during a one-second interval. An increase in FEVor FEV1 reflects a decrease in bronchoconstriction and an improvement inpulmonary function.

Forced Vital Capacity (FVC): The volume of air resulting from the forcedexpiratory flow test in which a subject first inspires maximally to thetotal lung capacity, then exhales as rapidly and as completely aspossible. An increase in FVC reflects a decrease in bronchoconstrictionand an improvement in pulmonary function.

Functional fragments, derivatives and variants of a polypeptide:Includes those fragments and variants that maintain one or morefunctions of the parent polypeptide. It is recognized that the gene orcDNA encoding a polypeptide can be considerably mutated withoutmaterially altering one or more the polypeptide's functions. First, thegenetic code is well known to be degenerate, and thus different codonsencode the same amino acids. Second, even where an amino acidsubstitution is introduced, the mutation can be conservative and have nomaterial impact on the essential functions of a protein. See Stryer,Biochemistry 3rd Ed., 1988. Third, part of a polypeptide chain can bedeleted without impairing or eliminating all of its functions. Fourth,insertions or additions can be made in the polypeptide chain, forexample, adding epitope tags—without impairing or eliminating itsfunctions (Ausubel et al., J. Immunol. 159:2502, 1997). Othermodifications that can be made without materially impairing one or morefunctions of a polypeptide include, for example, in vivo or in vitrochemical and biochemical modifications or which incorporate unusualamino acids. Such modifications include, for example, acetylation,carboxylation, phosphorylation, glycosylation, ubiquination, labeling,e.g., with radionucleotides, and various enzymatic modifications, aswill be readily appreciated by those well skilled in the art. A varietyof methods for labeling polypeptides and of substituents or labelsuseful for such purposes are well known in the art, and includeradioactive isotopes such as ³²P, ligands which bind to labeledantiligands (e.g., antibodies), fluorophores, chemiluminescent agents,enzymes, and antiligands. Functional fragments and variants can be ofvarying length. For example, some fragments have at least 10, 25, 50,75, 100, or 200 amino acid residues.

A functional fragment or variant of TSLP is a polypeptide which binds tothe TSLP receptor and induces a biological activity of TSLP, asdescribed in U.S. Pat. No. 6,555,520, which is incorporated herein byreference. It includes any polypeptide twenty or more amino acidresidues in length. Examples of functional fragments of TSLP include anN-terminal hydrophobic region that functions as a signal polypeptide, orthe cytoplasmic domain. Additional examples are amino acids 29-159 ofSEQ ID NO: 1, and a fragment having an N-terminus at amino acid 35 and aC-terminus at amino acid 159. Variants include amino acid sequences thatare at least about 80% identical to SEQ ID NO: 1, such as a polypeptideat least 90%, 95%, 97%, 98%, or 99% identical to SEQ ID NO: 1.Additional variants of TSLP include those resulting from alternativesplicing, and proteins in which differences in amino acid sequence areattributable to genetic polymorphism (allelic variation in individualsexpressing the protein). Other derivatives include fusion proteins, suchas those including poly-histidine sequence or an identifiablepolypeptide tag, and different glycosylation patterns. These andadditional fragments, variants and derivatives of TSLP are disclosed inU.S. Pat. No. 6,555,520, which is incorporated by reference herein.

γ_(c) (common cytokine receptor gamma chain): A receptor subunit thathas been shown to function as an essential signal-transducing componentof various cytokine receptors, including receptors for IL-4, IL-7, IL-9,IL-15 and IL-21. The γ_(c) subunit, together with a ligand-specificsubunit, forms high-affinity receptors for the respective cytokine.

Gene: A DNA sequence that comprises control and coding sequencesnecessary for the production of a polypeptide or protein. Thepolypeptide can be encoded by a full-length coding sequence or by anyportion of the coding sequence in some embodiments, so long as at leasta portion of the desired activity of the polypeptide is retained. A“foreign” or “heterologous” gene sequence is any nucleic acid that isintroduced into the genome of an animal by experimental manipulations.This can include gene sequences found in that animal so long as theintroduced gene contains some modification (e.g., a point mutation, thepresence of a selectable marker gene, a non-native regulatory sequence,or a native sequence integrated into the genome at a non-nativelocation, etc.) relative to the naturally-occurring gene.

Inflammatory lung disease: Many diseases of the lung are associated withlung inflammation. For example, ARDS is the rapid onset of progressivemalfunction of the lungs, and is usually associated with the malfunctionof other organs due to the inability to take up oxygen. The condition isassociated with extensive lung inflammation and small blood vesselinjury in all affected organs. ARDS is commonly precipitated by trauma,sepsis (systemic infection), diffuse pneumonia and shock. It may beassociated with extensive surgery, and certain blood abnormalities.

In many inflammatory lung diseases, the inflammatory response thataccompanies the underlying disease state is much more dangerous than theunderlying infection or trauma. Inflammatory lung diseases can include,but are not limited to pneumonia, ARDS, respiratory distress ofprematurity, chronic bronchitis, COPD, cystic fibrosis, pulmonaryfibrosis, and pulmonary sarcoidosis. Asthma can be an inflammatory lungdisease, but does not necessarily have an inflammatory component.

Inflammatory response: An accumulation of white blood cells, eithersystemically or locally at the site of inflammation. The inflammatoryresponse may be measured by many methods well known in the art, such asthe number of white blood cells (WBC), the number of polymorphonuclearneutophils (PMN) and/or neutrophils, a measure of the degree of PMNactivation, such as luminal enhanced-chemiluminescence, or a measure ofthe amount of cytokines present. Asthma can include an inflammatoryresponse including for example, increased numbers and/or PMNs,eosinophils or mast cells.

Inspiratory Flow Rate: The rate at which air travels into the lungsduring inspiration. Inspiratory flow is measured by a simple pulmonarytest; in performing the test the subject takes as deep and rapid abreath as possible from a machine known as a spirometer, which measuresthe rate of inspiration. An increase in inspiratory flow rate reflects adecrease in bronchoconstriction and an improvement in pulmonaryfunction.

Interleukin-7 (IL-7): Murine IL-7 is a glycoprotein of 25 kDa thatcontains six cysteine residues. It is derived from a precursor proteincontaining a classical secretory signal sequence of 25 amino acids. Thedisulfide bonds are essential for the biological activity of theprotein. Human IL-7 includes 152 amino acids and has a molecular weightof 17.4 kDa. The human protein is 17 amino acids longer than the murineprotein (the human gene contains an additional exon). Human and murineIL-7 (129 amino acids) have 60 percent sequence identity at the proteinlevel. The human IL-7 gene maps to chromosome 8q12-q13, has a length ofapproximately 33 kb, and contains six exons. The murine IL-7 gene mapsto chromosome 3, has a length of approximately 56 kb and contains fiveexons. IL-7 plays a role in both B and T cell proliferation.

Isolated: An “isolated” biological component (such as a nucleic acidmolecule, protein or organelle) has been substantially separated orpurified away from other biological components in the cell of theorganism in which the component naturally occurs, i.e., otherchromosomal and extra-chromosomal DNA and RNA, proteins and organelles.Nucleic acids and proteins that have been “isolated” include nucleicacids and proteins purified by standard purification methods. The termalso embraces nucleic acids and proteins prepared by recombinantexpression in a host cell as well as chemically synthesized nucleicacids.

Leukotriene Antagonist/Leukotriene Formation Inhibitor: Drugs that blockthe effects of leukotrienes (leukotriene antagonists) or inhibit theformation of leukotrienes (leukotriene formation inhibitors).Leukotrienes are substances that are associated with an allergicresponse and inflammation. In the airways, they cause bronchial oralveolar narrowing and increase secretions. Drugs can interfere withleukotriene action by inhibiting their synthesis (for example, zileuton,ZYFLO®, Abbott Laboratories) or blocking the receptor to which they bind(for example, monteleukast, SINGULAIR®, Merck and Company, and others).

Lung Volume: The maximum volume the lungs can hold.

Lymphocytes: A type of white blood cell that is involved in the immunedefenses of the body. There are two main types of lymphocytes: B cellsand T cells. Natural Killer Cell: A large granular lymphocyte capable ofkilling a tumor or microbial cell without prior exposure to the targetcell and without having it presented with or marked by ahistocompatibility antigen.

Neutralizing amount: An amount of an agent sufficient to decrease theactivity or amount of a substance to a level that is undetectable usingstandard method.

Nucleotide: “Nucleotide” includes, but is not limited to, a monomer thatincludes a base linked to a sugar, such as a pyrimidine, purine orsynthetic analogs thereof, or a base linked to an amino acid, as in apeptide nucleic acid (PNA). A nucleotide is one monomer in apolynucleotide. A nucleotide sequence refers to the sequence of bases ina polynucleotide.

Oligonucleotide: An oligonucleotide is a plurality of joined nucleotidesjoined by native phosphodiester bonds, between about 6 and about 300nucleotides in length. An oligonucleotide analog refers to moieties thatfunction similarly to oligonucleotides but have non-naturally occurringportions. For example, oligonucleotide analogs can contain non-naturallyoccurring portions, such as altered sugar moieties or inter-sugarlinkages, such as a phosphorothioate oligodeoxynucleotide. Functionalanalogs of naturally occurring polynucleotides can bind to RNA or DNA,and include peptide nucleic acid (PNA) molecules.

Particular oligonucleotides and oligonucleotide analogs can includelinear sequences up to about 200 nucleotides in length, for example asequence (such as DNA or RNA) that is at least 6 bases, for example atleast 8, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100 or even 200 bases long,or from about 6 to about 50 bases, for example about 10-25 bases, suchas 12, 15 or 20 bases.

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein-coding regions, in the samereading frame.

Open reading frame: A series of nucleotide triplets (codons) coding foramino acids without any internal termination codons. These sequences areusually translatable into a peptide.

Pharmaceutical agent: A chemical compound or composition capable ofinducing a desired therapeutic or prophylactic effect when properlyadministered to a subject or a cell. “Incubating” includes a sufficientamount of time for a drug to interact with a cell. “Contacting” includesincubating a drug in solid or in liquid form with a cell. An “anti-viralagent” or “anti-viral drug” is an agent that specifically inhibits avirus from replicating or infecting cells. Similarly, an“anti-retroviral agent” is an agent that specifically inhibits aretrovirus from replicating or infecting cells.

A “therapeutically effective amount” is a quantity of a chemicalcomposition or an anti-viral agent sufficient to achieve a desiredeffect in a subject being treated. For instance, this can be the amountnecessary to inhibit viral replication or to measurably alter outwardsymptoms of the viral infection, such as increase of T cell counts inthe case of an HIV infection. In general, this amount will be sufficientto measurably inhibit virus (e.g., HIV) replication or infectivity. Whenadministered to a subject, a dosage will generally be used that willachieve target tissue concentrations (for example, in lymphocytes) thathas been shown to achieve in vitro inhibition of viral replication.

Pharmaceutically acceptable carriers (excipients): The pharmaceuticallyacceptable carriers useful with the methods described herein areconventional. Remington's Pharmaceutical Sciences, by E. W. Martin, MackPublishing Co., Easton, Pa., 15th Edition (1975), describes compositionsand formulations suitable for pharmaceutical delivery of the cytokinesand cells disclosed herein.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Polypeptide: A polymer in which the monomers are amino acid residuesthat are joined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used, the L-isomers being preferred in nature. The termpolypeptide or protein as used herein encompasses any amino acidsequence and includes, but may not be limited to, modified sequencessuch as ADP-ribosylated proteins, ribosyl-proteins, and glycoproteins.The term polypeptide is specifically intended to cover naturallyoccurring proteins, such as TSLP, as well as polypeptides (for example,TSLP or a fragment thereof) that are recombinantly or syntheticallyproduced.

Substantially purified polypeptide as used herein refers to apolypeptide that is substantially free of other proteins, lipids,carbohydrates or other materials with which it is naturally associated.In one embodiment, the polypeptide is at least 50%, for example at least80% free of other proteins, lipids, carbohydrates or other materialswith which it is naturally associated. In another embodiment, thepolypeptide is at least 90% free of other proteins, lipids,carbohydrates or other materials with which it is naturally associated.In yet another embodiment, the polypeptide is at least 95% free of otherproteins, lipids, carbohydrates or other materials with which it isnaturally associated.

Probes and primers: Nucleic acid probes and primers can be readilyprepared based on a nucleic acid sequence. A probe comprises an isolatednucleic acid attached to a detectable label or reporter molecule.Typical labels include radioactive isotopes, enzyme substrates,co-factors, ligands, chemiluminescent or fluorescent agents, haptens,and enzymes. Methods for labeling and guidance in the choice of labelsappropriate for various purposes are discussed, e.g., in Sambrook etal., in Molecular Cloning: A Laboratory Manual, CSHL, New York, 1989,and Ausubel et al., in Current Protocols in Molecular Biology, GreenePubl. Assoc. and Wiley-Intersciences, 1992.

Primers are short nucleic acid molecules, preferably DNAoligonucleotides 10 nucleotides or more in length. More preferably,longer DNA oligonucleotides can be about 15, 17, 20 or 23 nucleotides ormore in length. Primers can be annealed to a complementary target DNAstrand by nucleic acid hybridization to form a hybrid between the primerand the target DNA strand, and then the primer extended along the targetDNA strand by a DNA polymerase enzyme. Primer pairs can be used foramplification of a nucleic acid sequence, e.g., by the polymerase chainreaction (PCR) or other nucleic-acid amplification methods known in theart.

Methods for preparing and using probes and primers are described, forexample, in Sambrook et al., in Molecular Cloning: A Laboratory Manual,CSHL, New York, 1989; Ausubel et al., in Current Protocols in MolecularBiology, Greene Publ. Assoc. and Wiley-Intersciences, 1998; and Innis etal., PCR Protocols, A Guide to Methods and Applications, Academic Press,Inc., San Diego, Calif., 1990. PCR primer pairs can be derived from aknown sequence, for example, by using computer programs intended forthat purpose such as Primer (Version 0.5, © 1991, Whitehead Institutefor Biomedical Research, Cambridge, Mass.). One of ordinary skill in theart will appreciate that the specificity of a particular probe or primerincreases with its length. Thus, for example, a primer comprising 30consecutive nucleotides of the TSLP encoding nucleotide will anneal to atarget sequence, such as another nucleic acid encoding TSLP, with ahigher specificity than a corresponding primer of only 15 nucleotides.Thus, in order to obtain greater specificity, probes and primers can beselected that comprise at least 17, 20, 23, 25, 30, 35, 40, 45, 50 ormore consecutive nucleotides of the nucleotide sequence of interest.

Protein: A biological molecule expressed by a gene and comprised ofamino acids. Also termed “polypeptide.”

Pulmonary function: The function of the respiratory system, which can bemeasured through a variety of tests, including, but not limited tomeasurements of airflow (e.g. spirometry) or arterial blood gases.Measurements of airflow included airflow rate, peak expiratory flow rate(PEFR), forced expiratory volume in the first second (FEV₁), and maximalmidexpiratory rate (MMEFR). A decrease in airflow rates throughout thevital capacity is the cardinal pulmonary function abnormality in asthma.Although essential for the diagnosis of asthma, it is not specific, asother obstructive diseases share this feature. The PEFR, FEV1, and MMEFRcan all be decreased in asthma. The severity of the attack of asthma canbe assessed by objective measurements of airflow.

Purified: The term “purified” does not require absolute purity; rather,it is intended as a relative term. Thus, for example, a purified proteinpreparation is one in which the protein referred to is more pure thanthe protein in its natural environment within a cell.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination can be accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, e.g., by genetic engineering techniques.

Respiratory Disorder: A large variety of abnormalities arising in allthe different structures of the body involved with gas exchange. Thesestructures include the lungs, nose, oropharynx, extrapulmonary airways,thoracic cage and respiratory muscles. Respiratory disorders encompassboth acute and chronic diseases. Asthma is one specific, non-limitingexample of a respiratory disorder. Other specific non-limiting examplesinclude, but are not limited to, coughs, pneumonia, bronchitis, such aschronic obstructive bronchitis, and emphysema, interstitial lungdisease, cystic fribrosis and lung tumors.

Rhino-conjunctivitis: A set of conditions that affect a subject'sconjunctiva and/or nasal membranes. Subjects affected withrhino-conjunctivitis include subjects that have rhinitis,conjunctivitis, and that symptoms associated with both conditions.

Allergic rhinitis is characterized by sneezing, rhinorrhea, obstructionof the nasal passages, conjunctival, nasal and pharengeal itching, andlacrimation. These symptoms all occur in a temporal relation to allergenexpose. The three forms of allergic rhinitis are

Allergic conjunctivitis is extremely common. Three forms are recognizedwith closely overlapping manifestations. Hay fever conjunctivitis has aseasonal incidence related to the release of airborne antigens. Vernalconjunctivitis is also seasonal, becoming worse in warm months. Atopicconjunctivitis occurs in subjects with atopic dermatitis and asthma.Airbourne antigens are associated with all three forms.

Sequence identity: The similarity between two nucleic acid sequences, ortwo amino acid sequences, is expressed in terms of the similaritybetween the sequences, otherwise referred to as sequence identity.Sequence identity is frequently measured in terms of percentage identity(or similarity or homology); the higher the percentage, the more similarthe two sequences are. Homologs or orthologs of the human CD4 protein,and the corresponding cDNA sequence, will possess a relatively highdegree of sequence identity when aligned using standard methods. Thishomology will be more significant when the orthologous proteins or cDNAsare derived from species which are more closely related (e.g., human andchimpanzee sequences), compared to species more distantly related (e.g.,human and murine sequences).

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-244, 1988; Higgins & Sharp,CABIOS 5:151-153, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90,1988; Huang et al., Computer Appls. in the Biosciences 8:155-65, 1992;and Pearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-410, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403-410, 1990) is available from several sources,including the National Center for Biotechnology Information (NCBI,Bethesda, Md.) and on the Internet, for use in connection with thesequence analysis programs blastp, blastn, blastx, tblastn and tblastx.It can be accessed at the NCBI website, together with a description ofhow to determine sequence identity using this program.

Homologs of the disclosed human CD4 protein typically possess at least60% sequence identity counted over full-length alignment with the aminoacid sequence of human CD4 using the NCBI Blast 2.0, gapped blastp setto default parameters. For comparisons of amino acid sequences ofgreater than about 30 amino acids, the Blast 2 sequences function isemployed using the default BLOSUM62 matrix set to default parameters,(gap existence cost of 11, and a per residue gap cost of 1). Whenaligning short peptides (fewer than around 30 amino acids), thealignment should be performed using the Blast 2 sequences function,employing the PAM30 matrix set to default parameters (open gap 9,extension gap 1 penalties). Proteins with even greater similarity to thereference sequence will show increasing percentage identities whenassessed by this method, such as at least 70%, at least 75%, at least80%, at least 90%, at least 95%, at least 98%, or at least 99% sequenceidentity. When less than the entire sequence is being compared forsequence identity, homologs will typically possess at least 75% sequenceidentity over short windows of 10-20 amino acids, and can possesssequence identities of at least 85% or at least 90% or 95% depending ontheir similarity to the reference sequence. Methods for determiningsequence identity over such short windows are described in the NCBIwebsite. These sequence identity ranges are provided for guidance only;it is entirely possible that strongly significant homologs could beobtained that fall outside of the ranges provided.

An alternative indication that two nucleic acid molecules are closelyrelated is that the two molecules hybridize to each other understringent conditions. Stringent conditions are sequence-dependent andare different under different environmental parameters. Generally,stringent conditions are selected to be about 5° C. to 20° C. lower thanthe thermal melting point (Tm) for the specific sequence at a definedionic strength and pH. The T_(m) is the temperature (under defined ionicstrength and pH) at which 50% of the target sequence remains hybridizedto a perfectly matched probe or complementary strand. Conditions fornucleic acid hybridization and calculation of stringencies can be foundin Sambrook et al., Molecular Cloning: A Laboratory Manual, CSHL, NewYork, 1989, and Tijssen, Laboratory Techniques in Biochemistry andMolecular Biology—Hybridization with Nucleic Acid Probes Part I, Chapter2, Elsevier, New York, 1993. Nucleic acid molecules that hybridize understringent conditions to a given sequence will typically hybridize underwash conditions of 2×SSC at 50° C.

Nucleic acid sequences that do not show a high degree of identity cannevertheless encode similar amino acid sequences, due to the degeneracyof the genetic code. It is understood that changes in nucleic acidsequence can be made using this degeneracy to produce multiple nucleicacid molecules that all encode substantially the same protein.

Selection markers or selectable markers: Refers to the use of a genethat encodes an enzymatic activity that confers resistance to anantibiotic or drug upon the cell in which the selectable marker isexpressed. Selectable markers can be “positive;” positive selectablemarkers typically are dominant selectable markers, i.e. genes thatencode an enzymatic activity that can be detected in any mammalian cellor cell line. Examples of dominant selectable markers include, but arenot limited to, (1) the bacterial aminoglycoside 3′ phosphotransferasegene (also referred to as the neo gene) which confers resistance to thedrug G418 in mammalian cells, (2) the bacterial hygromycin Gphosphotransferase (hyg) gene which confers resistance to the antibiotichygromycin and (3) the bacterial xanthine-guanine phosphoribosyltransferase gene (also referred to as the gpt gene) which confers theability to grow in the presence of mycophenolic acid. Selectable markerscan be “negative;” negative selectable markers encode an enzymaticactivity whose expression is cytotoxic to the cell when grown in anappropriate selective medium. For example, the HSV-tk gene and the dtgene are commonly used as a negative selectable marker. Expression ofthe HSV-tk gene in cells grown in the presence of gancyclovir oracyclovir is cytotoxic; thus, growth of cells in selective mediumcontaining gancyclovir or acyclovir selects against cells capable ofexpressing a functional HSV TK enzyme. Similarly, the expression of thedt gene selects against cells capable of expressing the Diphtheriatoxin. The terms are further defined, and methods further explained, byU.S. Pat. No. 5,464,764.

An animal whose genome “comprises a heterologous selectable marker gene”is an animal whose genome contains a selectable marker gene notnaturally found in the animal's genome that is introduced by means ofmolecular biological methods. A heterologous selectable marker isdistinguished from an endogenous gene naturally found in the animal'sgenome in that expression or activity of the heterologous selectablemarker can be selected for or against.

Small Molecule: The term small molecule encompasses a wide variety ofchemical compounds, including both inorganic and organic molecules. Bydefinition, a molecule is the smallest unit of matter that can exist byitself while retaining its chemical properties. A macromolecule is alarge molecule in which there is a large number of one or severalrelatively simple structural units, each consisting of several atomsbonded together, e.g., nucleic acids, polypeptides, polysaccharides. Inthe context of drug development, the term small molecule is used torefer to compounds that are not macromolecules (e.g., biologicalmacromolecules such as nucleic acids, proteins, polypeptides, etc.). Asmall molecule is typically made up of a single structural andfunctional unit or a small number of simple structural units. Smallmolecules are purified or isolated from natural products, or can beproduced synthetically. Frequently, small molecules are members oflibraries produced by combinatorial chemistry. Typically, a smallmolecule is less than about 1000 Daltons.

Specific binding agent: An agent that binds substantially only to adefined target. In one embodiment, the specific binding agent is amonoclonal or polyclonal antibody that specifically binds TSLP, or itsreceptor. Thus a TSLP receptor specific binding agent is an agent thatbinds substantially to a TSLP receptor.

The term “specifically binds” refers with respect to an antigen, such asthe TSLP or its receptor, to the preferential association of an antibodyor other ligand, in whole or part, with a cell or tissue bearing thatantigen and not to cells or tissues lacking that antigen. It is, ofcourse, recognized that a certain degree of non-specific interaction mayoccur between a molecule and a non-target cell or tissue. Nevertheless,specific binding may be distinguished as mediated through specificrecognition of the receptor or antigen. Although selectively reactiveantibodies bind antigen, they may do so with low affinity. On the otherhand, specific binding results in a much stronger association betweenthe antibody (or other ligand) and cells bearing the antigen thanbetween the bound antibody (or other ligand) and cells lacking theantigen. Specific binding typically results in greater than 2-fold, suchas greater than 5-fold, greater than 10-fold, or greater than 100-foldincrease in amount of bound antibody or other ligand (per unit time) toTSLP, or a cell or tissue bearing TSLP receptor as compared to adifferent polypeptide or to a cell or tissue lacking TSLP receptor,respectively. Specific binding to a protein under such conditionsrequires an antibody that is selected for its specificity for aparticular protein. A variety of immunoassay formats are appropriate forselecting antibodies or other ligands specifically immunoreactive with aparticular protein. For example, solid-phase ELISA immunoassays areroutinely used to select monoclonal antibodies specificallyimmunoreactive with a protein. See Harlow & Lane, ANTIBODIES, ALABORATORY MANUAL, Cold Spring Harbor Publications, New York (1988), fora description of immunoassay formats and conditions that can be used todetermine specific immunoreactivity.

T Cell: A white blood cell critical to the immune response. T cellsinclude, but are not limited to, CD4⁺ T cells and CD8⁺ T cells. A CD4⁺ Tlymphocyte is an immune cell that carries a marker on its surface knownas “cluster of differentiation 4” (CD4). These cells, also commonlyknown as helper T cells, help orchestrate the immune response, includingantibody responses as well as killer T cell responses. CD8⁺ T cellscarry the “cluster of differentiation 8” (CD8) marker. In oneembodiment, a CD8 T cell is a cytotoxic T lymphocytes. In anotherembodiment, a CD8 cell is a suppressor T cell.

Thymic Stromal Lymphopoietin (TSLP): A growth factor that binds aspecific receptor and stimulates lymphocyte proliferation. TSLP is knownto play a role in B cell development and maturation. Amino acidsequences of TSLP, functional variants, derivatives and fragmentsthereof, are provided in U.S. Pat. No. 6,555,520, which is incorporatedherein by reference. Nucleic acid sequences encoding these polypeptidesare also provided in U.S. Pat. No. 6,555,520.

TSLP has been shown to induce activation and phosphorylation of STAT-3and STAT-5 but does not activate any of the four known Janus kinases.TSLP mediated activation of STAT-5 can be uncoupled from cellproliferation.

The TSLP receptor has been shown to be a member of the hematopoietinreceptor superfamily. The receptor has been cloned and the murinecounterpart of which has been identified as delta-1. High affinitybinding sites require the presence of the alpha chain of the IL-7receptor. The receptor complex does not involve common gamma (γ_(c)), asignal-transducing component of various cytokine receptors, includingreceptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21.

Treatment: Refers to both prophylactic inhibition of initial infection,and therapeutic interventions to alter the natural course of anuntreated disease process, such as infection with a virus (e.g., HIVinfection).

Transformed: A transformed cell is a cell into which has been introduceda nucleic acid molecule by molecular biology techniques. As used herein,the term transformation encompasses all techniques by which a nucleicacid molecule might be introduced into such a cell, includingtransfection with viral vectors, transformation with plasmid vectors,and introduction of DNA by electroporation, lipofection, and particlegun acceleration.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. Recombinant DNA vectors are vectorshaving recombinant DNA. A vector can include nucleic acid sequences thatpermit it to replicate in a host cell, such as an origin of replication.A vector can also include one or more selectable marker genes and othergenetic elements known in the art. Viral vectors are recombinant DNAvectors having at least some nucleic acid sequences derived from one ormore viruses.

Virus: Microscopic infectious organism that reproduces inside livingcells. A virus consists essentially of a core of a single nucleic acidsurrounded by a protein coat, and has the ability to replicate onlyinside a living cell. “Viral replication” is the production ofadditional virus by the occurrence of at least one viral life cycle. Avirus may subvert the host cells' normal functions, causing the cell tobehave in a manner determined by the virus. For example, a viralinfection may result in a cell producing a cytokine, or responding to acytokine, when the uninfected cell does not normally do so.

“Retroviruses” are RNA viruses wherein the viral genome is RNA. When ahost cell is infected with a retrovirus, the genomic RNA is reversetranscribed into a DNA intermediate which is integrated very efficientlyinto the chromosomal DNA of infected cells. The integrated DNAintermediate is referred to as a provirus. The term “lentivirus” is usedin its conventional sense to describe a genus of viruses containingreverse transcriptase. The lentiviruses include the “immunodeficiencyviruses” which include human immunodeficiency virus (HIV) type 1 andtype 2 (HIV-1 and HIV-2), simian immunodeficiency virus (SIV), andfeline immunodeficiency virus (FIV).

HIV is a retrovirus that causes immunosuppression in humans (HIVdisease), and leads to a disease complex known as the acquiredimmunodeficiency syndrome (AIDS). “HIV disease” refers to awell-recognized constellation of signs and symptoms (including thedevelopment of opportunistic infections) in persons who are infected byan HIV virus, as determined by antibody or Western blot studies.Laboratory findings associated with this disease are a progressivedecline in T-helper cells.

The treatment of HIV disease has been significantly advanced by therecognition that combining different drugs with specific activitiesagainst different biochemical functions of the virus can help reduce therapid development of drug resistant viruses that were seen in responseto single drug treatment. In addition, discontinuation of existingtherapies results in a rapid rebound of viral replication, indicatingthe lack of complete HIV eradication by the drugs. There is therefore acontinuing need for the development of new anti-retroviral drugs thatact specifically at different steps of the viral infection andreplication cycle.

Wild-type: The term “wild-type” refers to a gene or gene product whichhas the characteristics of that gene or gene product when isolated froma naturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designatedthe “normal” or “wild-type” form of the gene. In contrast, the term“modified” or “mutant” refers to a gene or gene product that displaysmodifications in sequence and/or functional properties (i.e. alteredcharacteristics) when compared to the wild-type gene or gene product. Itis noted that naturally-occurring mutants can be isolated; these aretypically identified by the fact that they have altered characteristicswhen compared to the wild-type gene or gene product.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”All publications, patent applications, patents and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods and examplesare illustrative only and not intended to be limiting.

Methods for Inducing Proliferation of CD4⁺ T Cells

A method is provided herein for inducing the proliferation of CD4⁺ Tcells. In one embodiment, the method includes isolating CD4⁺ T cells andcontacting them with a therapeutically effective amount of TSLP, or aTSLP receptor agonist, thereby inducing proliferation of the CD4⁺ Tcells.

Amino acid sequences of TSLP, functional variants, derivatives andfragments thereof, are provided in U.S. Pat. No. 6,555,520, which isincorporated herein by reference. Nucleic acid sequences encoding thesepolypeptides are also provided in U.S. Pat. No. 6,555,520, which isincorporated by reference. In one specific example, a full-length TSLPpolypeptide is human TSLP and has an amino acid sequence set forth as:mfpfallyvl svsfrkifil qlvglvltyd ftncdfekik aaylstiskd (SEQ ID NO: 1)litymsgtks tefnntvscs nrphclteiq sltfnptagc aslakemfam ktkaalaiwcpgysetqina tqamkkrrkr kvttnkcleq vsqlqqlwrr fnrpllkqq

In another example, a full-length TSLP polypeptide is a murinepolypeptide has an amino acid sequence set forth as: mvllrslfilqvlvrmglty nfsncnftsi tkiycniifh dltgdlkgak feqiedcesk pacllkieyytlnpipgcps realndhcpg ypeterndgt qemaqevqni clnqtsqilr lwysfmqspe(mouse, TSLP, GenBank Accession No. NP_607342, SEQ ID NO: 2) ormravtwaiva mllprvlgai ptrtprtggv gdtlsvaivc hdlesvevtw gpgsahhglsanlslefryg nqvpqpcphy flldsvragc vlpmgkglle vvlregggak lfsrkkkasawlrprppwnv tlswvgdtva vscpshsypg leyevqhrdd fdpewqstsa pfcnltvggldpgrcydfrv ratpqdfyyg pearpskwtg vaslqgvgpt gsctgptlpr tpgtptpplalacglavall tlvlllallr mrrvkeallp gvpdprgsfp glfekhhgnf qawiadsqaavptvpeqdkd ddvirpqtkg vetqedddvi apgspclggg almsvggasf lmgdsgyttl (ratTSLP, GenBank Accession No. NP_604460, SEQ ID NO: 3)

Additional TSLP polypeptides have the following amino acid sequence:mktkaalaiw cpgysetqin atqamkkrrk rkvttnkcle qvsqlqglwr rfnrpllkqq (humanisoform 2, GenBank Accession No. AAH16720, SEQ ID NO: 4)

Another TSLP polypeptide has the following amino acid sequence:mkclgqskke evsfrkifil qlvglvltyd ftncdfekik aaylstiskd litymsgtkstefnntvscs nrphclteiq sltfnptagc aslakemfam ktkaalaiwc pgysetqinatqamkkrrkr kvttnkcleq vsqlqglwrr fnrpllkqq (GenBank Accession No.AAH40592, SEQ ID NO: 5)

The polypeptide set forth as SEQ ID NO: 1 includes an N-terminalhydrophobic region (amino acids 1-28) that functions as a signalpolypeptide, and a cleavage signal at amino acid 34. Thus, functionalfragments of SEQ ID NO: 1 include amino acids 29-159 of SEQ ID NO: 1 andamino acids 35-159 of SEQ ID NO: 1. In addition, functional polypeptidesinclude fusion polypeptides including poly-histidine, an antigenicepitope or a FLAG polypeptide, the sequences of which are set forth inU.S. Pat. No. 6,555,520, which is herein incorporated by reference.

A method is provided herein for stimulating T cell proliferation. Themethod includes contacting isolated mammalian CD4⁺ T cells with aneffective amount of a thymic stromal derived lymphopoietin (TSLP)polypeptide.

In one specific, non-limiting example, the population of T cellsincludes CD4⁺ T cells as greater than 50% of the population, greaterthan 80% of the population, greater than 90% of the population, greaterthan 95% of the population, or greater than 99% of the population. Avariety of methods can be used in order to purify CD4⁺ T cells.

In one embodiment, suspension of cells is produced, and antibodies thatspecifically bind CD4 are reacted with the cells in suspension. Methodsof determining the presence or absence of a cell surface marker, such asCD4, are well known in the art. Typically, labeled antibodiesspecifically directed to the marker are used to identify the cellpopulation. The antibodies can be conjugated to other compoundsincluding, but not limited to, enzymes, magnetic beads, colloidalmagnetic beads, haptens, fluorochromes, metal compounds, radioactivecompounds or drugs. The enzymes that can be conjugated to the antibodiesinclude, but are not limited to, alkaline phosphatase, peroxidase,urease and 3-galactosidase. The fluorochromes that can be conjugated tothe antibodies include, but are not limited to, fluoresceinisothiocyanate, tetramethylrhodamine isothiocyanate, phycoerythrin,allophycocyanins and Texas Red. For additional fluorochromes that can beconjugated to antibodies, see Haugland, R. P., Molecular Probes:Handbook of Fluorescent Probes and Research Chemicals (1992-1994). Themetal compounds that can be conjugated to the antibodies include, butare not limited to, ferritin, colloidal gold, and particularly,colloidal superparamagnetic beads. The haptens that can be conjugated tothe antibodies include, but are not limited to, biotin, digoxigenin,oxazalone, and nitrophenol. The radioactive compounds that can beconjugated or incorporated into the antibodies are known to the art, andinclude but are not limited to technetium 99m (⁹⁹ Tc), ¹²⁵I and aminoacids comprising any radionucleotides, including, but not limited to,¹⁴C, ³H and ³⁵S.

Fluorescence activated cell sorting (FACS) can be used to sort cellsthat express CD4, by contacting the cells with an appropriately labeledantibody. In one embodiment, additional antibodies and FACS sorting canfurther be used to produce substantially purified populations of CD4⁺ Tcells.

A FACS employs a plurality of color channels, low angle and obtuselight-scattering detection channels, and impedance channels, among othermore sophisticated levels of detection, to separate or sort cells. AnyFACS technique may be employed as long as it is not detrimental to theviability of the desired cells. (For exemplary methods of FACS, see U.S.Pat. No. 5,061,620, herein incorporated by reference).

However, other techniques of differing efficacy may be employed topurify and isolate desired populations of cells. The separationtechniques employed should maximize the retention of viability of thefraction of the cells to be collected. The particular technique employedwill, of course, depend upon the efficiency of separation, cytotoxicityof the method, the ease and speed of separation, and what equipmentand/or technical skill is required.

Separation procedures can include magnetic separation, usingantibody-coated magnetic beads, affinity chromatography, cytotoxicagents, either joined to a monoclonal antibody or used in conjunctionwith complement, and “panning,” which utilizes a monoclonal antibodyattached to a solid matrix, or another convenient technique. Antibodiesattached to magnetic beads and other solid matrices, such as agarosebeads, polystyrene beads, hollow fiber membranes and plastic petridishes, allow for direct separation. Cells that are bound by theantibody can be removed from the cell suspension by simply physicallyseparating the solid support from the cell suspension. The exactconditions and duration of incubation of the cells with the solidphase-linked antibodies will depend upon several factors specific to thesystem employed. The selection of appropriate conditions, however, iswell within the skill in the art.

The unbound cells then can be eluted or washed away with physiologicbuffer after sufficient time has been allowed for the cells expressing amarker of interest (e.g. CD4) to bind to the solid-phase linkedantibodies. The bound cells are then separated from the solid phase byany appropriate method, depending mainly upon the nature of the solidphase and the antibody employed.

Antibodies may be conjugated to biotin, which then can be removed withavidin or streptavidin bound to a support, or fluorochromes, which canbe used with a fluorescence activated cell sorter (FACS), to enable cellseparation (see above).

Polynucleotides encoding TSLP are also of use in the methods disclosedherein (see U.S. Pat. No. 6,555,520, herein incorporated by reference).For example, one such polynucleotide includes (SEQ ID NO: 11):   1GCAGCCAGAA AGCTCTGGAG CATCAGGGAG ACTCCAACTT AAGGCAACAG  51 CATGGGTGAATAAGGGCTTC CTGTGGACTG GCAATGAGAG GCAAAACCTG 101 GTGCTTGAGC ACTGGCCCCTAAGGCAGGCC TTACAGATCT CTTACACTCG 151 TGGTGGGAAG AGTTTAGTGT GAAACTGGGGTGGAATTGGG TGTCCACGTA 201 TGTTCCCTTT TGCCTTACTA TATGTTCTGT CAGTTTCTTTCAGGAAAATC 251 TTCATCTTAC AACTTGTAGG GCTGGTGTTA ACTTACGACT TCACTAACTG301 TGACTTTGAG AAGATTAAAG CAGCCTATCT CAGTACTATT TCTAAAGACC 351TGATTACATA TATGAGTGGG ACCAAAAGTA CCGAGTTCAA CAACACCGTC 401 TCTTGTAGCAATCGGCCACA TTGCCTTACT GAAATCCAGA GCCTAACCTT 451 CAATCCCACC GCCGGCTGCGCGTCGCTCGC CAAAGAAATG TTCGCCATGA 501 AAACTAAGGC TGCCTTAGCT ATCTGGTGCCCAGGCTATTC GGAAACTCAG 551 ATAAATGCTA CTCAGGCAAT GAAGAAGAGG AGAAAAAGGAAAGTCACAAC 601 CAATAAATGT CTGGAACAAG TGTCACAATT ACAAGGATTG TGGCGTCGCT651 TCAATCGACC TTTACTGAAA CAACAGTAAA CCATCTTTAT TATGGTCATA 701TTTCACAGCC CAAAATAAAT CATCTTTATT AAGTAAAAAA AAA

One of skill in the art, using the genetic code, can readily identifydegenerate variants of SEQ ID NO: 11, which also encode TSLP.Polynucleotides include DNA, cDNA and RNA sequences which encode a TSLP.It is understood that all polynucleotides encoding TSLP are alsoincluded herein, as long as they encode a polypeptide that has anactivity of TSLP, such as the ability to induce proliferation of a CD4⁺T cell. Such polynucleotides include naturally occurring, synthetic, andintentionally manipulated polynucleotides. For example, a polynucleotideencoding may be subjected to site-directed mutagenesis. Thepolynucleotides include sequences that are degenerate as a result of thegenetic code, but encode TSLP. There are 20 natural amino acids, most ofwhich are specified by more than one codon. Therefore, all degeneratenucleotide sequences are of use in the methods disclosed herein as longas the amino acid sequence of the TSLP encoded by the nucleotidesequence is functionally unchanged.

DNA sequences encoding TSLP can be expressed in vitro (or in vivo) byDNA transfer into a suitable host cell. Methods of stable transfer,meaning that the foreign DNA is continuously maintained in the host, areknown in the art. Polynucleotide sequences encoding TSLP can be insertedinto an expression vector, such as a plasmid, virus or other vehicleknown in the art that has been manipulated by insertion or incorporationof the TSLP sequences. Polynucleotide sequences which encode TSLP can beoperatively linked to expression control sequences. In one embodiment,an expression control sequence operatively linked to a coding sequenceis ligated such that expression of the coding sequence is achieved underconditions compatible with the expression control sequences.

The polynucleotide encoding TSLP, such as human TSLP, can be insertedinto an expression vector that contains a promoter sequence whichfacilitates the efficient transcription of the inserted genetic sequenceby the host. The expression vector typically contains an origin ofreplication, a promoter, as well as specific genes that allow phenotypicselection of the transformed cells. Vectors suitable for use include,but are not limited to, the T7-based expression vector for expression inbacteria (Rosenberg et al., Gene 56:125, 1987), the pMSXND expressionvector for expression in mammalian cells (Lee and Nathans, J. Biol.Chem. 263:3521, 1988) and baculovirus-derived vectors for expression ininsect cells. The DNA segment can be present in the vector operablylinked to regulatory elements, for example, a promoter (e.g., T7,metallothionein I, or polyhedron promoters).

Polynucleotide sequences encoding TSLP can be expressed in eitherprokaryotes or eukaryotes. Hosts can include microbial, yeast, insectand mammalian organisms. Methods of expressing DNA sequences havingeukaryotic or viral sequences in prokaryotes are well known in the art.For example, biologically functional viral and plasmid DNA vectorscapable of expression and replication in a host are known in the art.Such vectors are used to incorporate a DNA sequence encoding TSLP.Transfection of a host cell with recombinant DNA can be carried out byconventional techniques and are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ methodusing procedures well known in the art. Alternatively, MgCl₂ or RbCl canbe used. Transformation can also be performed after forming a protoplastof the host cell if desired, or by electroporation.

When the host is a eukaryote, methods of transfection of DNA as calciumphosphate co-precipitates, conventional mechanical procedures such asmicroinjection, electroporation, insertion of a plasmid encased inliposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with a second foreign DNA molecule encoding a selectablephenotype, such as the herpes simplex thymidine kinase gene. Anothermethod is to use a eukaryotic viral vector, such as simian virus 40(SV40) or bovine papilloma virus, to transiently infect or transformeukaryotic cells and express the protein (see for example, EukaryoticViral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).

Methods of Treating an Immunodeficiency

The present methods also include administration of a therapeuticallyeffective amount of a TSLP polypeptide disclosed herein, a TSLP receptoragonist, or a nucleic acid encoding TSLP to treat an immunodeficiency,or a disease process in which increased numbers, proliferation, orfunction of CD4⁺ T cells is desired. In one embodiment, the disease is aviral disease, such as infection with an immunodeficiency virus. In oneexample, the immunodeficiency virus is a human immunodeficiency virus(HIV), such as HIV-1 or HIV-2. In another example, the subject has animmunodeficiency as a result of a genetic disorder, such as severecombined immunodeficiency (SCID). In a further embodiment, the subjecthas acquired the immunodeficiency as a result of an environmentalexposure or administration of an agent. For example, the agent can beradiation or a chemotherapeutic agent. Generally, the administration ofthe TSLP polypeptide, TSLP receptor agonist, or nucleic acid encodingTSLP reduces a sign or a symptom of the disorder. For example, theadministration can result in increased number or function of CD4⁺ cells.

A TSLP polypeptide or TSLP agonist can be included in a pharmaceuticalformulation in an amount per unit dose sufficient to evoke proliferationof CD4⁺ cells in the subject to be treated. The response can be thereduction of any other detrimental effect of the disease (e.g.,reduction in HIV protease activity, see U.S. Pat. No. 5,171,662),regardless of whether the protection is partial or complete. Thecomposition can be administered to the subject by any suitable means.Examples are by oral administration, intramuscular injection,subcutaneous injection, intravenous injection, intraperitonealinjection, eye drop or by nasal spray.

The dose of a TSLP polypeptide or other TSLP receptor agonist can varyaccording to factors such as the disease state, age, sex, immune status,and weight of the individual, and the ability to elicit a desiredresponse in the individual. Dosage regime may be adjusted to provide theoptimum therapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the therapeutic situation. In therapeutic applications,compositions are administered to a subject having a disorder in atherapeutically effective amount, which is an amount sufficient to cureor at least partially arrest the disease or a sign or symptom of thedisorder. Amounts effective for this use will depend upon the severityof the disorder and the general state of the patient's health. Aneffective amount of the compound is that which provides eithersubjective relief of a symptom(s) or an objectively identifiableimprovement as noted by the clinician or other qualified observer.

A TSLP polypeptide or a TSLP receptor agonist can be administered by anymeans known to one of skill in the art (see Banga, “ParenteralControlled Delivery of Therapeutic Peptides and Proteins,” inTherapeutic Peptides and Proteins, Technomic Publishing Co., Inc.,Lancaster, Pa., 1995) such as by intramuscular, subcutaneous, orintravenous injection, but even oral, nasal or anal administration iscontemplated. In one embodiment, administration is by subcutaneous orintramuscular injection. To extend the time during which the peptide orprotein is available to stimulate a response, a peptide or protein canbe provided as an implant, an oily injection, or as a particulatesystem. The particulate system can be a microparticle, a microcapsule, amicrosphere, a nanocapsule, or similar particle. (see, e.g., Banga,supra). A particulate carrier based on a synthetic polymer has beenshown to act as an adjuvant to enhance the immune response, in additionto providing a controlled release.

Thus, examples of compositions include liquid preparations for orifice(e.g., oral, nasal, anal, vaginal, peroral, intragastric) administrationsuch as suspensions, syrups or elixirs; and preparations for parenteral,subcutaneous, intradermal, intramuscular or intravenous administration(e.g., injectable administration, including the use of needlelessinjectors) such as sterile suspensions or emulsions, are provided. Insuch compositions the antigen(s) may be in admixture with a suitablecarrier, diluent, or excipient such as sterile water, physiologicalsaline, glucose or the like. The compositions can contain auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,adjuvants, gelling or viscosity enhancing additives, preservatives,flavoring agents, colors, and the like, depending upon the route ofadministration and the preparation desired. Standard texts, such asRemington's Pharmaceutical Science, 17th edition, 1985, incorporatedherein by reference, may be consulted to prepare suitable preparations,without undue experimentation. The compositions can also be lyophilized.

Suitable dosages can also be determined by one of skill in the art. Forexample, typical dosages of a TSLP polypeptide can be from about 5 μg/mlto about 150 μg/ml, and other dosages can be from about 15 to about 150μg/dose. Single or multiple administrations of the compositions areadministered depending on the dosage and frequency as required andtolerated by the subject. In one embodiment, the dosage is administeredonce as a bolus, but in another embodiment can be applied periodicallyuntil either a therapeutic result is achieved. Generally, the dose issufficient to treat or ameliorate symptoms or signs of disease withoutproducing unacceptable toxicity to the subject.

In another embodiment, a nucleic acid encoding a TSLP polypeptide isutilized (e.g., see Robinsion et al., Nat. Med., 5(5):526-34, 1999).Thus, a method is provided for treating a viral infection, such as anHIV infection or an immunodeficiency, by providing a therapeuticallyeffective amount of a nucleic acid encoding the TSLP polypeptide.Delivery of the polynucleotide encoding the CD4 fusion polypeptide canbe achieved using a recombinant expression vector such as a chimericvirus or a colloidal dispersion system, or through the use of targetedliposomes. For example, about 10 μg to about 1 mg of DNA can beutilized, such as about 10-100 μg, or about 50 μg, of a DNA constructcan be injected intradermally three times at two week intervals toproduce the desired therapeutic effect

Various viral vectors which can be utilized for administration ofnucleic acids include, but are not limited to, adenoviral, herpes viral,or retroviral vectors. In one embodiment, a retroviral vector such as aderivative of a murine or avian retroviral vector is utilized. Examplesof retroviral vectors in which a single foreign gene can be insertedinclude, but are not limited to: Moloney murine leukemia virus (MoMuLV),Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus(MuMTV), and Rous Sarcoma Virus (RSV). In addition, when the subject isa human, a vector such as the gibbon ape leukemia virus (GaLV) isutilized. A number of additional retroviral vectors can incorporatemultiple genes. The vectors can transfer or incorporate a gene for aselectable marker so that transduced cells can be identified andgenerated. By inserting a nucleic acid encoding a TSLP polypeptide intothe viral vector, along with another gene which encodes the ligand for areceptor on a specific target cell, for example, the vector is renderedtarget specific. Retroviral vectors can be made target specific byattaching, for example, a sugar, a glycolipid, or a protein. Targetingcan also be accomplished by using an antibody to target the retroviralvector. Those of skill in the art will know of, or can readily ascertainwithout undue experimentation, specific polynucleotide sequences whichcan be inserted into the retroviral genome or attached to a viralenvelope to allow target specific delivery of the retroviral vector.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. These plasmids are missing anucleotide sequence that enables the packaging mechanism to recognize anRNA transcript for encapsidation. Helper cell lines which have deletionsof the packaging signal include, but are not limited to, Q2, PA317 andPA12, for example. These cell lines produce empty virions, since nogenome is packaged. If a retroviral vector is introduced into such cellsin which the packaging signal is intact, but the structural genes arereplaced by other genes of interest, the vector can be packaged andvector virion produced.

Alternatively, NIH 3T3 or other tissue culture cells can be directlytransfected with plasmids encoding the retroviral structural genes gag,pol and env, by conventional calcium phosphate transfection. These cellsare then transfected with the vector plasmid containing the genes ofinterest. The resulting cells release the retroviral vector into theculture medium.

Another targeted delivery system for therapeutic TSLP polynucleotides isa colloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. Liposomes are artificial membrane vesicles thatare useful as delivery vehicles in vitro and in vivo. It has been shownthat large unilamellar vesicles (LUV), which range in size from 0.2-4.0μm, can encapsulate a substantial percentage of an aqueous buffercontaining large macromolecules. RNA, DNA and intact virions can beencapsulated within the aqueous interior and be delivered to cells in abiologically active form (Fraley et al., Trends Biochem. Sci. 6:77,1981). In order for a liposome to be an efficient gene transfer vehicle,the following characteristics should be present: (1) encapsulation ofthe genes of interest at high efficiency while not compromising theirbiological activity; (2) preferential and substantial binding to atarget cell in comparison to non-target cells; (3) delivery of theaqueous contents of the vesicle to the target cell cytoplasm at highefficiency; and (4) accurate and effective expression of geneticinformation (Mannino et al., Biotechniques 6:682, 1988; see also U.S.Pat. No. 6,270,795).

The composition of the liposome is usually a combination ofphospholipids, particularly high-phase-transition-temperaturephospholipids, usually in combination with steroids, such ascholesterol. Other phospholipids or other lipids may also be used. Thephysical characteristics of liposomes depend on pH, ionic strength, andthe presence of divalent cations. Examples of lipids useful in liposomeproduction include phosphatidyl compounds, such as phosphatidylglycerol,phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,sphingolipids, cerebrosides, and gangliosides. Particularly useful arediacylphosphatidyl-glycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

The targeting of liposomes can be classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The surface of the targeted delivery system may be modified in a varietyof ways. In the case of a liposomal targeted delivery system, lipidgroups can be incorporated into the lipid bilayer of the liposome inorder to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand.

In embodiments wherein treatment of viral disease is desired, such astreatment with an infection with an immunodeficiency virus, theadministration of a TSLP polypeptide, a TSLP receptor agonist, or anucleic acid encoding TSLP, can be combined with administration of oneor more anti-viral drugs useful in the treatment of viral disease. Forexample, a TSLP polypeptide, TSLP receptor agonist, or a nucleic acidencoding TSLP, can be administered, whether before or after exposure tothe virus, in combination with effective doses of other anti-virals,immunomodulators, anti-infectives, or vaccines. The term“administration” refers to both concurrent and sequential administrationof the active agents.

It should be noted that pharamaceutical compounds including atherapeutically effective amounts of TSLP antagonists can also beprepared using the information provided.

In one embodiment, a combination of TSLP, or a nucleic aid encoding TSLPwith one or more agents useful in the treatment of a viral disease isprovided. In one specific, non-limiting example, the viral disease is aretroviral disease, such as an HIV-1-induced, an HIV-2-induced, aSIV-induced, or a FIV induced disease. Examples of anti-virals that canbe used in the disclosed method are: AL-721 (from Ethigen of LosAngeles, Calif.), recombinant human interferon beta (from TritonBiosciences of Alameda, Calif.), Acemannan (from Carrington Labs ofIrving, Tex.), gangiclovir (from Syntex of Palo Alto, Calif.),didehydrodeoxythymidine or d4T (from Bristol-Myers-Squibb), EL10 (fromElan Corp. of Gainesville, Ga.), dideoxycytidine or ddC (fromHoffmann-LaRoche), Novapren (from Novaferon Labs, Inc. of Akron, Ohio),zidovudine or AZT (from Burroughs Wellcome), ribavirin (from Viratek ofCosta Mesa, Calif.), alpha interferon and acyclovir (from BurroughsWellcome), Indinavir (from Merck & Co.), 3TC (from Glaxo Wellcome),Ritonavir (from Abbott), Saquinavir (from Hoffmann-LaRoche), and others.

Examples of immuno-modulators are AS-101 (Wyeth-Ayerst Labs.),bropirimine (Upjohn), gamma interferon (Genentech), GM-CSF (GeneticsInstitute), IL-2 (Cetus or Hoffmann-LaRoche), human immune globulin(Cutter Biological), IMREG (from Imreg of New Orleans, La.), SK&F106528,TNF (Genentech), and soluble TNF receptors (Immunex).

Examples of some anti-infectives used include clindamycin withprimaquine (from Upjohn, for the treatment of pneumocystis pneumonia),fluconazlone (from Pfizer for the treatment of cryptococcal meningitisor candidiasis), nystatin, pentamidine, trimethaprim-sulfamethoxazole,and many others.

“Highly active anti-retroviral therapy” or “HAART” refers to acombination of drugs which, when administered in combination, inhibits aretrovirus from replicating or infecting cells better than any of thedrugs individually. In one embodiment, the retrovirus is a humanimmunodeficiency virus. In one embodiment, the highly activeanti-retroviral therapy includes the administration of3′axido-3-deoxy-thymidine (AZT) in combination with other agents.Examples of agents that can be used in combination in HAART for a humanimmunodeficiency virus are nucleoside analog reverse transcriptaseinhibitor drugs (NA), non-nucleoside analog reverse transcriptaseinhibitor drugs (NNRTI), and protease inhibitor drugs (PI). Onespecific, non-limiting example of HAART used to suppress an HIVinfection is a combination of indinavir and efavirenz, an experimentalnon-nucleoside reverse transcriptase inhibitor (NNRTI).

In one embodiment, HAART is a combination of three drugs used for thetreatment of an HIV infection, such as the drugs shown in Table 2 below.Examples of three-drug HAART for the treatment of an HIV infectioninclude 1 protease inhibitor from column A plus 2 nucleoside analogsfrom column B in Table 2. In addition, ritonavir and saquinavir can beused in combination with 1 or 2 nucleoside analogs. TABLE 2 Column AColumn B indinavir (Crixivan) AZT/ddI nelfinavir (Viracept) d4T/ddIritonavir (Norvir) AZT/ddC saquinavir (Fortovase) AZT/3TCritonavir/saquinavir d4T/3TC

In addition, other 3- and 4-drug combinations can reduce HIV to very lowlevels for sustained periods. The combination therapies are not limitedto the above examples, but include any effective combination of agentsfor the treatment of HIV disease (including treatment of AIDS).Administration of these agents is thus combined with the administrationof TSLP, or a nucleic acid encoding TSLP.

In another embodiment a method is provided for treating animmunodeficiency by isolating CD4⁺ T cells, contacting the CD4⁺ T cellswith an effective amount of a TSLP polypeptide, TSLP receptor agonist,or a nucleic acid encoding TSLP, and administering them to a subject.The CD4⁺ T cells can be autologous (from the same subject) orheterologous (from a different subject). In one embodiment, the subjectis human.

Fluorescence activated cell sorting (FACS) can be used to sort (isolate)cells CD4⁺ T cells, by contacting the cells with an appropriatelylabeled antibody. In one embodiment, several antibodies (such asantibodies that bind CD4, CD3, and/or CD8) and FACS sorting can be usedto produce substantially purified populations of CD4⁺ T cells. Thesemethods are known in the art.

As noted above, FACS employs a plurality of color channels, low angleand obtuse light-scattering detection channels, and impedance channels,among other more sophisticated levels of detection, to separate or sortcells. Any FACS technique may be employed as long as it is notdetrimental to the viability of the desired cells. (For exemplarymethods of FACS, see U.S. Pat. No. 5,061,620).

However, other techniques of differing efficacy may be employed topurify and isolate desired populations of cells. The separationtechniques employed should maximize the retention of viability of thefraction of the cells to be collected. The particular technique employedwill, of course, depend upon the efficiency of separation, cytotoxicityof the method, the ease and speed of separation, and what equipmentand/or technical skill is required.

Separation procedures may include magnetic separation, usingantibody-coated magnetic beads, affinity chromatography, cytotoxicagents, either joined to a monoclonal antibody or used in conjunctionwith complement, and “panning,” which utilizes a monoclonal antibodyattached to a solid matrix, or another convenient technique (see above).Antibodies attached to magnetic beads and other solid matrices, such asagarose beads, polystyrene beads, hollow fiber membranes and plasticpetri dishes, allow for direct separation. Cells that are bound by theantibody can be removed from the cell suspension by simply physicallyseparating the solid support from the cell suspension. The exactconditions and duration of incubation of the cells with the solidphase-linked antibodies will depend upon several factors specific to thesystem employed. The selection of appropriate conditions, however, iswell within the skill in the art.

The unbound cells then can be eluted or washed away with physiologicbuffer after sufficient time has been allowed for the cells expressing amarker of interest (e.g. CD4) to bind to the solid-phase linkedantibodies. The bound cells are then separated from the solid phase byany appropriate method, depending mainly upon the nature of the solidphase and the antibody employed. Antibodies may be conjugated to biotin,which then can be removed with avidin or streptavidin bound to asupport, or fluorochromes, which can be used with a fluorescenceactivated cell sorter (FACS), to enable cell separation.

For example, cells expressing CD4 are initially separated from othercells by the cell-surface expression of CD4. In one specific,non-limiting example, CD4⁺ cells are positively selected by magneticbead separation, wherein magnetic beads are coated with CD4 reactivemonoclonal antibody. The CD4⁺ cells are then removed from the magneticbeads.

Release of the CD4⁺ T cells from the magnetic beads can effected byculture release or other methods. Purity of the isolated CD4⁺ cells isthen checked with a FACSCAN.RTM. flow cytometer (Becton Dickinson, SanJose, Calif.), for example, if so desired. In one embodiment, furtherpurification steps are performed, such as FACS sorting the population ofcells released from the magnetic beads. In one example, this sorting canbe performed to detect expression of CD3 and/or CD8. The purified CD4⁺ Tcells are then contacted with a therapeutically effective amount ofTSLP, or a nucleic acid encoding TSLP. Subsequently, the expanded CD4⁺ Tcells are then administered to the subject.

In one embodiment, CD4⁺ T cells are also contacted with an antigen. Theantigen can be any polypeptide of interest, including a viral antigen, abacterial antigen or a fungal antigen.

Examples of viruses include: Retroviridae (for example, humanimmunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III,LAV or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP;Picornaviridae (for example, polio viruses, hepatitis A virus;enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses);Calciviridae (such as strains that cause gastroenteritis); Togaviridae(for example, equine encephalitis viruses, rubella viruses); Flaviridae(for example, dengue viruses, encephalitis viruses, yellow feverviruses); Coronaviridae (for example, coronaviruses); Rhabdoviridae (forexample, vesicular stomatitis viruses, rabies viruses); Filoviridae (forexample, ebola viruses); Paramyxoviridae (for example, parainfluenzaviruses, mumps virus, measles virus, respiratory syncytial virus);Orthomyxoviridae (for example, influenza viruses); Bungaviridae (forexample, Hantaan viruses, bunga viruses, phleboviruses and Nairoviruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g.,reoviruses, orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae(Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae(papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses);Herpesviridae (herpes simplex virus (HSV) 1 and HSV-2, varicella zostervirus, cytomegalovirus (CMV), herpes viruses); Poxyiridae (variolaviruses, vaccinia viruses, pox viruses); and Iridoviridae (such asAfrican swine fever virus); and unclassified viruses (for example, theetiological agents of Spongiform encephalopathies, the agent of deltahepatities (thought to be a defective satellite of hepatitis B virus),the agents of non-A, non-B Hepatitis (class 1=internally transmitted;class 2=parenterally transmitted (i.e., Hepatitis C); Norwalk andrelated viruses, and astroviruses). Examples of bacteria include:Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia,Mycobacteria sps (such as M. tuberculosis, M. avium, M. intracellulare,M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae,Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes(Group A Streptococcus), Streptococcus agalactiae (Group BStreptococcus), Streptococcus (viridans group), Streptococcus faecalis,Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcuspneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilusinfluenzae, Bacillus antracis, corynebacterium diphtheriae,corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridiumperfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiellapneumoniae, Pasturella multocida, Bacteroides sp., Fusobacteriumnucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponemapertenue, Leptospira, and Actinomyces israelli. Examples of fungiinclude, but are not limited to, Cryptococcus neoformans, Histoplasmacapsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydiatrachomatis, Candida albicans. Other infectious organisms (such asprotists) include: Plasmodium falciparum and Toxoplasma gondii.

In one embodiment, isolated CD4⁺ T cells are contacted with an effectiveamount of TSLP, and are also contacted with an antigen. The antigen canbe any polypeptide of interest, including any viral antigen, bacterialantigen, or fungal antigen, such as an antigen from one of theinfectious organisms listed above.

Methods of Treating an Inflammatory Disorder

Methods are provided herein for treating an inflammatory disorder, suchas (but not limited to) asthma. The method includes administering to asubject a therapeutically effective amount of a TSLP antagonist. Methodsare also provided herein for treating immune-mediated disorders of thelung, such as asthma. In one example, the disorder is asthma, whereinthe asthma is antigen-mediated. Methods are also disclosed for thetreatment of an inflammatory disorder such as allergic rhinitis,allergic dermatitis, and allergic conjunctivitis. In one embodiment, theinflammatory disorder is an IgE-mediated disorder, such as a pulmonaryIgE mediated disorder. For example, the disorder can be asthma or thedisorder can be rhino-conjunctivitis.

Asthma (sometimes referred to as reactive airway disease) is a conditionof the respiratory tract characterized by reversible narrowing of theairways (bronchoconstriction) and increased sensitivity(hyperresponsiveness) of the airways to a variety of stimuli. Thefamiliar symptoms of asthma (such as coughing, wheezing, chesttightness, dyspnea) is caused by airway smooth muscle contraction,increased bronchial mucus secretion, and inflammation. Asthma has beenestimated to affect 10-20% of school-aged children around the world, andhospital admissions for asthma in children have increased dramaticallyin recent years, one survey for the United States indicating thathospital admissions for children under 15 with asthma increased by atleast 145% between 1970 and 1984 (See, Sears, in Asthma as anInflammatory Disease, O'Byrne, (ed.), Marcel Dekker, Inc.; New York,1990, pp. 15-48). Overall, it is estimated that 10 million Americans (4%of the population) have asthma, and even more than a decade ago about $4billion was spent in treatment per year (Altman, New York Times, TheDoctor's World, Mar. 26, 1991).

The inflammatory response in asthma is typical for tissues covered by amucosa and is characterized by vasodilation, plasma exudation,recruitment of inflammatory cells such as neutrophils, monocytes,macrophages, lymphocytes and eosinophils to the sites of inflammation,and release of inflammatory mediators by resident tissue cells (e.g.,mast cells) or by migrating inflammatory cells. In allergen-inducedasthma, sufferers often exhibit a dual response to exposure to anallergen—an “early phase” response beginning immediately after exposureand lasting until 1-2 hours after exposure, followed by a “late phase”response beginning about 3 hours after exposure and lasting sometimesuntil 8-10 hours or longer after exposure. Late phase response inallergen-induced asthma and persistent hyperresponsiveness have beenassociated with the recruitment of leukocytes, and particularlyeosinophils, to inflamed lung tissue.

The causes of asthma are not completely understood, however the study ofagents that trigger acute asthmatic episodes supports the theory thatasthma is an immunological reaction by a subject in response to specificallergens of the subject's environment (extrinsic asthma). These“triggers” exacerbate asthma by causing transient enhancement of airwayhyperresponsiveness. Triggers that have been found to induce airwayhyperresponsiveness include but are not limited to, inhaled allergens,inhaled low molecular weight agents to which the subject has becomesensitized (such as by previous exposure), viral or mycoplasmarespiratory infections. The common feature of inducing triggers is thatthey are associated with airway inflammation (see, Cockcroft, in Asthmaas an Inflammatory Disease, O'Byrne (ed.), Marcel Dekker, Inc.; NewYork, 1990, pp. 103-125).

Without being bound by theory, it is believed that, following exposureto a trigger, such as an allergin, antigen presenting cells (APCs), Tcells, B cells, eosinophils, mast cells, and basophils, contribute tothe mechanism of asthma. Specifically, the APCs present antigen to Tcells which, in turn, provoke B cells to produce IgE. B cells arestimulated to produce IgE by two types of signals, IL-4 or IL-13, anddirect contact from T cells (Barnes and Lemanske, New Engl. J. Med.,344:350-362, 2002). The released IgE activates mast cells which, inturn, cause constriction of the airways.

In order to treat or prevent an IgE-mediated disorder, such as asthma orrhino-conjunctivitis, a therapeutically effective amount of anantagonist of TSLP is administered to the subject. Generally, thisadministration results in the amelioration of a sign or a symptom of thedisorder.

In one embodiment an additional anti-infective agent, anti-inflammatoryagent, bronchodilator, enzyme, expectorant, leukotriene antagonist,leukotriene formation inhibitor, or mast cell stabilizer is administeredin conjunction with a TSLP antagonist. The administration of theadditional agent and the TSLP antagonist can be sequential orsimultaneous.

TSLP antagonists include small molecule antagonists, antibodies to TSLP,antibodies to the TSLP receptor, and TSLP receptor fusion proteins, suchas TSLPR-immunoglobulin Fc molecules or polypeptides that encodecomponents of more than one receptor chain, that thereby mimic aphysiological receptor heterodimer or higher order oligomer, amongstothers. TSLP has been shown to bind directly to a type I cytokinereceptor superfamily member (which are also known as hematopoietinreceptor superfamily members), TSLPR. TSLPR has been cloned. Thefunctional high-affinity receptor for TSLP has been demonstrated toinclude two polypeptides, TSLPR and the IL-7 receptor alpha chain. Thus,both TSLP and IL-7 shares IL-7Ralpha as a component of their receptors.However, these receptors are distinctive in that the TSLP receptoradditionally contains TSLPR whereas the IL-7 receptor additionallycontains the common cytokine receptor gamma chain, which is asignal-transducing component of various cytokine receptors. TSLPR (andFc fusions of this receptor chain) are described, for example, inPublished U.S. Patent Application No. 2002/0160949, which isincorporated herein by reference.

Interference with the ligand-receptor interaction has proven to be aneffective strategy for the development of antagonists. In oneembodiment, a ligand mutein of TSLP is utilized which retains receptorbinding activity, but fails to induce receptor signaling. In oneexample, the mutein is a TSLP polypeptide that binds the receptor butdoes not trigger signaling through the receptor. Alternatively, theantagonist can be a small molecule which interferes with the binding ofTSLP to its receptor. Small molecule libraries may be screened forcompounds which may block the interaction or signaling mediated by anidentified ligand-receptor pairing.

In another embodiment, a TSLP antagonist is an antibody thatspecifically binds to a specified cytokine ligand, such as TSLP (forexample, primate, human, cat, dog, rat or mouse TSLP). In anotherembodiment, a TSLP antagonist is an antibody that binds the TSLPreceptor.

A number of immunogens may be selected to produce antibodiesspecifically reactive with ligand or receptor proteins. Recombinantprotein, such as a recombinant TSLP polypeptide or a TSLP receptorpolypeptide can be used for the production of monoclonal or polyclonalantibodies. Naturally occurring protein, can also be used either in pureor partially purified form. Synthetic peptides, made using theappropriate protein sequences, can also be used as an immunogen for theproduction of antibodies. Recombinant protein can be expressed andpurified in eukaryotic or prokaryotic cells (see Coligan, et al. (eds.),Current Protocols in Protein Science, John Wiley and Sons, New York,N.Y., 1995; and Ausubel, et al., Current Protocols in Molecular Biology,Greene/Wiley, New York, N.Y., 1987). Naturally folded or denaturedmaterial can be used, as appropriate, for producing antibodies. Eithermonoclonal or polyclonal antibodies may be generated.

Methods of producing polyclonal antibodies are well known to those ofskill in the art. Typically, an immunogen, preferably a purifiedprotein, is mixed with an adjuvant and animals are immunized with themixture. The animal's immune response to the immunogen preparation ismonitored by taking test bleeds and determining the titer of reactivityto the protein of interest. For example, when appropriately high titersof antibody to the immunogen are obtained, usually after repeatedimmunizations, blood is collected from the animal and antisera areprepared. Further fractionation of the antisera to enrich for antibodiesreactive to the protein can be performed if desired. Immunization canalso be performed through other methods, e.g., DNA vector immunization(see, for example, Wang, et al. Virology 228:278-284, 1997).

Monoclonal antibodies can be obtained by various techniques familiar toresearchers skilled in the art. Typically, spleen cells from an animalimmunized with a desired antigen are immortalized, commonly by fusionwith a myeloma cell (for example, see, Kohler and Milstein, Eur. J.Immunol. 6:511-519, 1976). Alternative methods of immortalizationinclude transformation with Epstein Barr Virus, oncogenes, orretroviruses, or other methods known in the art (for example see Doyle,et al. (eds.) Cell and Tissue Culture: Laboratory Procedures, John Wileyand Sons, New York, N.Y., 1994). Colonies arising from singleimmortalized cells are screened for production of antibodies of thedesired specificity and affinity for the antigen, such as a TSLPpolypeptide or the TSLP receptor. Yield of the monoclonal antibodiesproduced by such cells may be enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate host.Alternatively, DNA sequences which encode a monoclonal antibody or abinding fragment thereof can be isolated by screening a DNA library fromhuman B cells.

Antibodies include intact molecules as well as fragments thereof, suchas Fab, F(ab′)₂, and Fv which include a heavy chain and light chainvariable region and are capable of binding the epitopic determinant.These antibody fragments retain some ability to selectively bind withtheir antigen or receptor and are defined as follows:

-   -   (1) Fab, the fragment which contains a monovalent        antigen-binding fragment of an antibody molecule, can be        produced by digestion of whole antibody with the enzyme papain        to yield an intact light chain and a portion of one heavy chain;    -   (2) Fab′, the fragment of an antibody molecule can be obtained        by treating whole antibody with pepsin, followed by reduction,        to yield an intact light chain and a portion of the heavy chain;        two Fab′ fragments are obtained per antibody molecule;    -   (3) (Fab′)₂, the fragment of the antibody that can be obtained        by treating whole antibody with the enzyme pepsin without        subsequent reduction; F(ab′)₂ is a dimer of two Fab′ fragments        held together by two disulfide bonds;    -   (4) Fv, a genetically engineered fragment containing the        variable region of the light chain and the variable region of        the heavy chain expressed as two chains; and    -   (5) Single chain antibody (such as scFv), defined as a        genetically engineered molecule containing the variable region        of the light chain, the variable region of the heavy chain,        linked by a suitable polypeptide linker as a genetically fused        single chain molecule.

Methods of making these fragments are known in the art (see for example,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, New York, 1988). An epitope is any antigenic determinant onan antigen to which the paratope of an antibody binds. Epitopicdeterminants usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three-dimensional structural characteristics, as well asspecific charge characteristics.

In one example, the variable region is an Fv, which includes thevariable region of the light chain and the variable region of the heavychain expressed as individual polypeptides. Fv antibodies are typicallyabout 25 kDa and contain a complete antigen-binding site with 3 CDRs pereach heavy chain and each light chain. The V_(H) and the V_(L) can beexpressed from two individual nucleic acid constructs. If the V_(H) andthe V_(L) are expressed non-contiguously, the chains of the Fv antibodyare typically held together by noncovalent interactions. However, thesechains tend to dissociate upon dilution, so methods have been developedto crosslink the chains through glutaraldehyde, intermoleculardisulfides, or a peptide linker. Thus, in one example, the Fv can be adisulfide stabilized Fv (dsFv), wherein the heavy chain variable regionand the light chain variable region are chemically linked by disulfidebonds.

One of skill in the art will realize that conservative variants of theantibodies can be produced. Such conservative variants employed in dsFvfragments or in scFv fragments will retain critical amino acid residuesnecessary for correct folding and stabilizing between the V_(H) and theV_(L) regions, and will retain the charge characteristics of theresidues in order to preserve the low pI and low toxicity of themolecules. Amino acid substitutions (such as at most one, at most two,at most three, at most four, or at most five amino acid substitutions)can be made in the V_(H) and the V_(L) regions to increase yield.

Antibody fragments can be prepared by proteolytic hydrolysis of theantibody or by expression in E. coli of DNA encoding the fragment.Antibody fragments can be obtained by pepsin or papain digestion ofwhole antibodies by conventional methods. For example, antibodyfragments can be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab′)₂. This fragment can befurther cleaved using a thiol reducing agent, and optionally a blockinggroup for the sulfhydryl groups resulting from cleavage of disulfidelinkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, anenzymatic cleavage using pepsin produces two monovalent Fab′ fragmentsand an Fc fragment directly (see U.S. Pat. No. 4,036,945 and U.S. Pat.No. 4,331,647, and references contained therein; Nisonhoff et al., Arch.Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959;Edelman et al., Methods in Enzymology, Vol. 1, page 422, Academic Press,1967.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

For example, Fv fragments comprise an association of V_(H) and V_(L)chains. This association may be noncovalent (Inbar et al., Proc. Nat'lAcad. Sci. U.S.A. 69:2659, 1972). Alternatively, the variable chains canbe linked by an intermolecular disulfide bond or cross-linked bychemicals such as glutaraldehyde. See, e.g., Sandhu, supra. Thus, a dsFvcan be produced. In an additional example, the Fv fragments compriseV_(H) and V_(L) chains connected by a peptide linker. These single-chainantigen binding proteins (sFv) are prepared by constructing a structuralgene comprising DNA sequences encoding the V_(H) and V_(L) domainsconnected by an oligonucleotide. The structural gene is inserted into anexpression vector, which is subsequently introduced into a host cellsuch as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are known in the art (see Whitlow et al.,Methods: a Companion to Methods in Enzymology, 2:97, 1991; Bird et al.,Science 242:423, 1988; U.S. Pat. No. 4,946,778; Pack et al.,Bio/Technology 11:1271, 1993; and Sandhu, supra).

Humanized monoclonal antibodies are produced by transferring donorantibody complementarity determining regions from heavy and lightvariable chains of the mouse immunoglobulin into a human variabledomain, and then substituting human residues in the framework regions ofthe donor counterparts. The use of antibody components derived fromhumanized monoclonal antibodies obviates potential problems associatedwith the immunogenicity of the constant regions of the donor antibody.Techniques for producing humanized monoclonal antibodies are described,for example, by Jones et al., Nature 321:522, 1986; Riechmann et al.,Nature 332:323, 1988; Verhoeyen et al., Science 239:1534, 1988; Carteret al., Proc. Nat'l Acad. Sci. U.S.A. 89:4285, 1992; Sandhu, Crit. Rev.Biotech. 12:437, 1992; and Singer et al., J. Immunol. 150:2844, 1993.

Antibodies to TSLP polypeptides are known in the art. In addition,anti-TSLPR antibodies are commercially available (R & D Systems,Minneapolis, Minn., cat. no. MAB981; DNAX Research, Inc., Palo Alto,Calif.). Antibodies are also prepared against TSLP receptor or TSLP byimmunization with specified epitopes, such as regions of increasedantigenicity determined by the Welling plot of Vector NTI.RTM. Suite(Informax, Inc, Bethesda, Md.). The sequence of the TSLP receptor, andregions of increased antigenicity in human TSLP receptor are disclosedin U.S. Patent Publication No. 2003/0186875. Pharmaceutical compositions(see above) generally include a therapeutically effective amount of aTSLP antagonist, and can also include additional agents. The preparationof pharmaceutical compositions is disclosed above.

An effective amount of a TSLP antagonist can be administered in a singledose, or in multiple doses, for example daily, during a course oftreatment. In one embodiment, a therapeutically effective amount of aTSLP antagonist is administered as a single pulse dose, as a bolus dose,or as pulse doses administered over time. Thus, in pulse doses, a bolusadministration of a TSLP antagonist is provided, followed by a timeperiod wherein no TSLP antagonist is administered to the subject,followed by a second bolus administration. In specific, non-limitingexamples, pulse doses of a TSLP antagonist are administered during thecourse of a day, during the course of a week, or during the course of amonth.

Thus, the TSLP antagonist disclosed herein may be administered to asubject for the treatment of an inflammatory disorder. The TSLPantagonist can be administered to subject to treat an IgE-mediateddisorder in a subject, such as asthma, in that individual. TSLPantagonist administration can be systemic or local. Local administrationof the TSLP anatagonist is performed by methods well known to thoseskilled in the art. By way of example, one method of administration tothe lungs of an individual is by inhalation through the use of anebulizer or inhaler. For example, the TSLP antagonist is formulated inan aerosol or particulate and drawn into the lungs using a standardnebulizer well known to those skilled in the art.

In other embodiments, the administration of the TSLP antagonist issystemic. Oral, intravenous, intraarterial, subcutaneous,intraperitoneal, intramuscular, and even rectal administration iscontemplated.

The effectiveness of treatment with a TSLP antagonist can be measured bymonitoring sign or symptoms of the IgE mediated disorder. For monitoringasthma, pulmonary function can be assessed by methods known to those ofskill in the art. For example, various measurable parameters of lungfunction can be studied before, during, or after treatment. Pulmonaryfunction can be monitored by testing any of several physicallymeasurable operations of a lung including, but not limited to,inspiratory flow rate, expiratory flow rate, and lung volume. Astatistically significant increase, as determined by mathematicalformulas well known to those skilled in the art, in one or more of theseparameters indicates efficacy of the TSLP antagonist treatment.

The methods of measuring pulmonary function most commonly employed inclinical practice involve timed measurement of inspiratory andexpiratory maneuvers to measure specific parameters. For example, FVCmeasures the total volume in liters exhaled by a patient forcefully froma deep initial inspiration. This parameter, when evaluated inconjunction with the FEV1, allows bronchoconstriction to bequantitatively evaluated. A statistically significant increase, asdetermined by mathematical formulas well known to those skilled in theart, in FVC or FEV 1 reflects a decrease in bronchoconstriction, andindicates that antagonist therapy is effective.

A problem with forced vital capacity determination is that the forcedvital capacity maneuver (i.e., forced exhalation from maximuminspiration to maximum expiration) is largely technique dependent. Inother words, a given subject may produce different FVC values during asequence of consecutive FVC maneuvers. The FEF 25-75 or forcedexpiratory flow determined over the midportion of a forced exhalationmaneuver tends to be less technique dependent than the FVC. Similarly,the FEV1 tends to be less technique-dependent than FVC. Thus, astatistically significant increase, as determined by mathematicalformulas well known to those skilled in the art, in the FEF 25-75 orFEV1 reflects a decrease in bronchoconstriction, and indicates that TSLPantagonist therapy is effective.

In addition to measuring volumes of exhaled air as indices of pulmonaryfunction, the flow in liters per minute measured over differing portionsof the expiratory cycle can be useful in determining the status of apatient's pulmonary function. In particular, the peak expiratory flow,taken as the highest airflow rate in liters per minute during a forcedmaximal exhalation, is well correlated with overall pulmonary functionin a patient with asthma and other respiratory diseases. Thus, astatistically significant increase, as determined by mathematicalformulas well known to those skilled in the art, in the peak expiratoryflow following administration of a TSLP antagonist indicates that thetherapy is effective.

Production of a Knock-Out Mouse

A transgenic mouse is disclosed herein whose somatic and germ cellscomprise a disrupted TSLP receptor gene, the disruption being sufficientto inhibit the binding of TSLP to a TSLP receptor (TSLP^(−/−)). Micethat are TSLP^(−/−) exhibit a diminished inflammatory response in thelungs in response to an antigen. Transgenic mice are also disclosedherein that include a disrupted TSLP receptor gene, and a disruptedγ_(C) gene, the disruption being sufficient to inhibit the production ofa receptor including γ_(C) or to disrupt the signaling of IL-7 throughthe IL-7 receptor. Mice that are TSLP receptor (R)^(−/−)γ^(−/−), or thatare TSLP^(−/−) γ_(C) ^(−/−), that have a decreased T cell activity arealso encompassed by this disclosure. One of skill in the art, using thedescription provided herein, can readily produce these animals.

A DNA molecule containing a desired gene sequence, such as a γ_(C) or aTSLP receptor gene sequence, can be introduced into pluripotent cells(such as ES cells) by any method that will permit the introducedmolecule to undergo recombination at its regions of homology. Techniquesthat can be used include calcium phosphate/DNA co-precipitates,microinjection of DNA into the nucleus, electroporation, bacterialprotoplast fusion with intact cells, transfection, polycations, e.g.,polybrene, polyornithine, etc. The DNA can be single or double strandedDNA, linear or circular. Techniques for transforming mammalian cells areknown, and examples for such methods are described, for instance, inKeown et al., Meth. Enzym. 185:527-537, 1990 and Mansour et al., Nature336:348-352, 1988.

Some methods, such as direct microinjection, or calcium phosphatetransformation, may cause the introduced nucleic acid molecule to formconcatemers upon integration. These concatemers can resolve themselvesto form non-concatemeric integration structures. An alternative methodfor introducing the gene to the pluripotent cell is electroporation(Toneguzzo, et al., Nucleic Acids Res. 16:5515-5532, 1988; Quillet etal., J. Immunol. 141:17-20,1988; Machy et al., Proc. Natl. Acad. Sci.USA 85:8027-8031, 1988).

After introduction of the DNA molecule(s), the cells are usuallycultured under conventional conditions, as are known in the art. Aselectable marker (as discussed above) can be used to facilitate therecovery of those cells that have received the DNA molecule containingthe desired gene sequence. For the purposes of the present disclosure,any gene sequence whose presence in a cell permits recognition andclonal isolation of the cell can be employed as a detectable marker,whether or not it conveys a survival advantage in the transgenic cell.

After selection for cells that have incorporated the desired DNAmolecule, the cells are cultured, and the presence of the introduced DNAmolecule is confirmed as described above. For instance, approximately10⁷ cells are cultured and screened for cells that have undergone asecond recombinational event (discussed below), resulting in thereplacement of a native sequence (i.e. a gene sequence that is normallyand naturally present in the recipient cell) with the desired genesequence. Any of a variety of methods can be used to identify cells thathave undergone the second recombinational event, including directscreening of clones, use of PCR, use of hybridization probes, etc.

In one embodiment, a gene is located upstream or downstream from thetargeting construct that provides for identification of whether a doublecrossover (and therefore targeted integration, not random integration)has occurred. By way of example, the herpes simplex virus thymidinekinase (HSV-tk) gene can be employed for this purpose, since thepresence of the thymidine kinase gene is detected by the use ofnucleoside analogs, such as acyclovir or gangcyclovir, for theircytotoxic effects on cells that contain a functional HSV-tk gene. Theabsence of sensitivity to these nucleoside analogs indicates the absenceof the thymidine kinase and indicates that, therefore, where homologousrecombination has occurred, a double crossover event has also occurred.

Once the DNA molecule containing the construct has been introduced intothe ES cells (or other pluripotent cells), the construct recombines withthe wild-type TSLP receptor or the γ_(C) gene through the process ofhomologous recombination. Homologous recombination provides a method forintroducing a desired gene sequence into a plant or animal cell andproducing chimeric or transgenic plants and animals having defined, andspecific, gene alterations. A discussion of the process of homologousrecombination can be found in Watson, J. D., In: Molecular Biology ofthe Gene, 3rd Ed., W. A. Benjamin, Inc., Menlo Park, Calif., 1977.

In brief, homologous recombination is a well-studied natural cellularprocess that results in the scission of two nucleic acid moleculeshaving identical or substantially similar sequences (i.e. “homologous”),and the ligation of the two molecules such that one region of eachmolecule initially present is now ligated to a region of the otherinitially present molecule (Sedivy, Bio-Technol. 6:1192-1196, 1988).Homologous recombination is a sequence-specific process by which cellscan transfer a “region” of DNA from one DNA molecule to another. As usedherein, a “region” of DNA is intended to generally refer to any nucleicacid molecule. The region can be of any length from a single base to asubstantial fragment of a chromosome, and can, but needs not, includecoding regions for one or more proteins.

For homologous recombination to occur between two DNA molecules, themolecules can possess a “region of homology” with respect to oneanother. Such a region of homology is usually at least two base pairslong, but is more customarily 2-20 Kb long. Two DNA molecules possesssuch a “region of homology” when one contains a region whose sequence isso similar to a region in the second molecule that base pairing andhomologous recombination can occur. Recombination is usually catalyzedby enzymes that are naturally present in both prokaryotic and eukaryoticcells.

The transfer of a region of DNA can be envisioned as occurring through amulti-step process. If either of the two participant nucleic acidmolecules is circular, then a recombination event results in theintegration of the circular molecule into the other participant nucleicacid molecule. Importantly, if a particular region is flanked on bothsides by regions of homology (which can be the same, but can also bedifferent), then two recombinational events can occur, thus resulting inthe exchange of a region of DNA between two DNA molecules. Recombinationcan be “reciprocal,” and thus result in an exchange of DNA regionsbetween two recombining DNA molecules. Alternatively, it can be“nonreciprocal” (also referred to as “gene conversion”) and result inboth recombining nucleic acid molecules having the same nucleotidesequence.

For homologous recombination, constructs are prepared where the gene ofinterest is flanked on one or both sides with DNA homologous with theDNA of the target region. The homologous DNA is generally within 100 Kb,but can be within 50 Kb, 25 Kb, or, in some embodiments, about 2.5 Kb or1.5 Kb or more of the target gene. The homologous DNA can include the5′-upstream region comprising any enhancer sequences, transcriptionalinitiation sequences, the region 5′ of these sequences, or the like. Thehomologous region can include a portion of the coding region, where thecoding region of a gene can include an open reading frame or combinationof exons and introns. The homologous region can comprise all or aportion of an intron, where all or a portion of one or more exons alsocan be present.

Alternatively, the homologous region can comprise the 3′-region, so asto comprise all or a portion of the transcription termination region ofa gene, or the region 3′ thereof. The homologous regions can extend overall or a portion of a target gene, or be outside the target gene butinclude all or a portion of the transcriptional regulatory regions ofthe structural gene. In many embodiments, the homologous sequence willbe joined to the gene of interest, proximally or distally. Usually, asequence other than the wild-type sequence normally associated with thetarget gene will be used to separate the homologous sequence from thegene of interest on at least one side of the gene of interest. Someportion of the sequence can be the 5′ or 3′ sequence associated with thegene of interest (the target).

In order to prepare the subject recombining constructs, it is necessaryto know the sequence that is targeted for homologous recombination.While a sequence of 14 bases complementary to a sequence in a genome canprovide for homologous recombination, normally the individual flankingsequences will be at least about 150 bp, and can be 12 Kb or more, butusually not more than about 8 Kb. The sizes of the flanking regions aredetermined by the size of the known sequence, the number of sequences inthe genome which can have homology to the site for integration, whethermutagenesis is involved and the extent of separation of the regions formutagenesis, the particular site for integration, or the like. Suitablemethods for homologous recombination are described, for example, in PCTPublication No. WO 02/14495 A2.

In one embodiment, a targeting vector is utilized for functionallydisrupting an endogenous γ_(C) or TSLP receptor gene in a cell includes:

-   -   a) a nonhomologous replacement portion;    -   b) a first homology region located upstream of the nonhomologous        replacement portion, the first homology region having a        nucleotide sequence with substantial identity to a first γ_(C)        or TSLP receptor gene sequence; and    -   c) a second homology region located downstream of the        nonhomologous replacement portion, the second homology region        having a nucleotide sequence with substantial identity to a        second γ_(C) or TSLP receptor gene sequence, the second γ_(C) or        TSLP receptor gene sequence having a location downstream of the        first γ_(C) or TSLP receptor gene sequence in a naturally        occurring endogenous γ_(C) or TSLP receptor gene.

Thus, the nonhomologous replacement portion is flanked 5′ and 3′ bynucleotide sequences with substantial identity to a γ_(C) or a TSLPreceptor gene sequences. A nucleotide sequence with “substantialidentity” to a γ_(C) or a TSLP receptor gene sequence is intended todescribe a nucleotide sequence having sufficient homology to a γ_(C) ora TSLP receptor gene sequence to allow for homologous recombinationbetween the nucleotide sequence and an endogenous γ_(C) or TSLP receptorgene sequence in a host cell. Typically, the nucleotide sequences of theflanking homology regions are at least 90%, more preferably at least95%, even more preferably at least 98% and most preferably 100%identical to the nucleotide sequences of the endogenous γ_(C) or TSLPreceptor gene to be targeted for homologous recombination. In oneembodiment, the flanking homology regions are isogenic with the targetedendogenous allele (e.g., the DNA of the flanking regions is isolatedfrom cells of the same genetic background as the cell into which thetargeting construct is to be introduced). Additionally, the flankinghomology regions of the targeting vector are of sufficient length forhomologous recombination between the targeting vector and an endogenousγ_(C) or a TSLP receptor gene in a host cell when the vector isintroduced into the host cell. Typically, the flanking homology regionsare at least 1 kilobase in length and more preferably are least severalkilobases in length.

Chimeric or transgenic animals are prepared, for example, by introducinga TSLP receptor or a γ_(C) construct, as described herein, into aprecursor pluripotent cell, such as an ES cell, or equivalent, asdescribed above, and in Robertson, E. J., in: Current Communications inMolecular Biology, Capecchi, M. R. (ed.), Cold Spring Harbor Press, ColdSpring Harbor, N.Y., 1989, pp. 39-44. The term “precursor” is intendedto denote only that the pluripotent cell is a precursor to the desired(“transfected”) pluripotent cell that is prepared in accordance with theteachings of the present disclosure. The pluripotent (precursor ortransfected) cell can be cultured in vivo, in a manner known in the art(Evans et al., Nature 292:154-156, 1981) to form a chimeric ortransgenic animal.

Any ES cell can be used in accordance with the present disclosure. Forinstance, in some embodiments, primary isolates of ES cells are used.Such isolates can be obtained directly from embryos such as the CCE cellline disclosed by Robertson, E. J., in: Current Communications inMolecular Biology, Capecchi, M. R. (ed.), Cold Spring Harbor Press, ColdSpring Harbor, N.Y., 1989, pp. 39-44), or from the clonal isolation ofES cells from the CCE cell line (Schwartzberg, et al., Science246:799-803, 1989). Such clonal isolation can be accomplished accordingto the method of E. J. Robertson (in: Teratocarcinomas and EmbryonicStem Cells: A Practical Approach (E. J. Robertson, Ed.), IRL Press,Oxford, 1987). The purpose of such clonal propagation is to obtain EScells that have a greater efficiency for differentiating into an animal.Examples of ES cell lines that have been clonally derived from embryosare the ES cell lines, AB 1 (hprt+) or AB2.1 (hprt⁻).

The ES cells can be cultured on stromal cells (such as STO cells(especially SNC4 STO cells) and/or primary embryonic fibroblast cells)as described by E. J. Robertson (in: Teratocarcinomas and Embryonic StemCells: A Practical Approach, (E. J. Robertson, Ed.), IRL Press, Oxford,1987, pp. 71-112). The stromal (and/or fibroblast) cells serve toeliminate the clonal overgrowth of abnormal ES cells. The cells can becultured in the presence of leukocyte inhibitory factor (“lif”) (Goughet al., Reprod. Fertil. Dev. 1:281-288, 1989; Yamamori et al., Science246:1412-1416, 1989). Since the gene encoding lif has been cloned (Goughet al., Reprod. Fertil. Dev. 1:281-288, 1989), it is also possible totransform stromal cells with this gene, by methods known in the art, andto then culture the ES cells on transformed stromal cells that secretelif into the culture medium.

ES cell lines can be derived or isolated from any species (for example,chicken, etc.), including cells derived or isolated from mammals such asrodents (i.e. mouse, rat, hamster, etc.), rabbits, sheep, goats, fish,pigs, cattle and primates such as humans. In one embodiment, the mammalis a mouse.

Transformed ES cells thereafter can be injected into blastocysts.Blastocysts containing the targeted ES cells are implanted intopseudo-pregnant females and allowed to develop to term. The ES cellsthereafter colonize the embryo and can contribute to the germ line ofthe resulting chimeric animal (Jaenisch, Science 240:1468-1474, 1988).Chimeric offspring are identified, for instance, by coat-color markers,and those showing chimerism are selected for breeding offspring. Thoseoffspring that carry the mutant allele can be identified by coat coloror other markers, and the presence of the mutant allele reaffirmed byDNA analysis of blood samples.

A “double knock-out” can be generated by introducing two constructs intoa single ES cell, one designed to undergo homologous recombination withthe endogenous (wild-type) γ_(C) gene (or another gene of interest, suchas Rag2), and one designed to undergo homologous recombination with theendogenous (wild-type) TSLP receptor gene. Alternatively, one line ofmice can be generated that are homozygous for a γ_(C) gene deletion(knock-out), or for the deletion of another gene of interest (forexample, Rag2, see below). An additional line of mice can be generatedthat are homozygous for a TSLP receptor gene deletion (knock-out). Thesetwo lines of mice can then be mated to produce mice that have both theother gene of interest (such as γ_(C) or Rag2) and the TSLP receptorgenes deleted.

In another embodiment, a line of mice can be generated that arehomozygous for a γ_(C) gene deletion. These animals are mated, andblastocysts are collected. Embryonic stem cells are then prepared fromthe γ_(C) ^(−/−) mice, and a construct is introduced into these cellsdesigned to undergo homologous recombination with the TSLP receptorgene. Resultant offspring are mated, and γ_(C) ^(−/−)TSLP receptor(R)^(−/−) mice are selected. In another embodiment, a line of mice canbe generated that are homozygous for a TSLP receptor gene deletion.These animals are mated, and blastocysts are collected from theTSLPR^(−/−) mice. Embryonic stem cells are then prepared from theTSLPR^(−/−) mice, and a construct is introduced into these cellsdesigned to undergo homologous recombination with the γ_(C) gene.Similar methods could be used to produce other double knock-out micehomozygous for a TSLP receptor deletion, and for a deletion of anothergene of interest, such as, but not limited to, Rag-2.

In addition to using homologous recombination in ES cells to producegene knock-outs, a recombining site/recombinase system can be used togenerate knock-outs gene (Rajewsky, J. Clin. Invest. 98:600-603, 1996).The Cre enzyme is a member of a large family of recombinases thatrecognizes specifically recombining sites (e.g. loxP, a sequence motifof 34 base pairs) and can induce recombination at these sites. If a DNAsegment is flanked by two loxP sites in the same orientation, Creexcises that segment from the DNA, leaving a single LoxP behind. Byappropriately positioning the two loxP sites, this system can be used togenerate deletions, such as a deletion in a TSLP receptor, TSLP or γ_(C)gene.

Thus, in one embodiment, a transgenic mouse is produced that includes aloxP flanked (floxed) gene, such as a TSLP receptor, TSLP or γ_(C) gene.Mice are also produced that include a promoter, such as a tissuespecific (e.g. an immunoglobulin promoter) or an inducible promoter(e.g. a temperature sensitive promoter), operably linked to Cre gene intheir genome. Mice including the floxed gene are then mated to the miceincluding the Cre gene. Under appropriate conditions, the expression ofCre is induced, and recombination occurs at the recombining sites,resulting in deletion of the floxed gene, such as the TSLP, TSLPreceptor or γ_(C) gene.

In one embodiment, γ_(C) ^(−/−)TSLPR^(−/−) mice exhibit decreasedcellularity of the thymus. In one embodiment, the cellularity of thethymus is decreased at least about 40%, 50%, 60%, 70%, 80%, 90%, 95%, or99% as compared to a control. In one embodiment, a control is a γ_(C)^(+/+)TSLPR^(+/+) (wild-type) animal. In another embodiment, a controlis a standard value. In another embodiment, γ_(C) ^(−/−)TSLPR^(−/−) miceexhibit decreased numbers of CD4⁺ T cells. Thus, the number of CD4⁺ Tcells is decreased by at least about 50%, such as at least about 60%,70%, 80%, 90%, 95%, or 99%, as compared to a control.

In an additional embodiment, TSLPR^(−/−) mice exhibit decreased responseto an antigen, such as an antigen known to induce an inflammatoryresponse, and/or induce the production of IgE. For example, TSLPR^(−/−)mice exhibit a decreased inflammatory response following administrationof ovalbumin (OVA).

In one embodiment, γ_(C) ^(−/−)TSLPR^(−/−) mice are used to test agentsdesigned to affect T cell proliferation. Thus, an agent is administeredto γ_(C) ^(−/−)TSLPR^(−/−) mice, and its effect on the number of CD4⁺ Tcells and/or the cellularity of the thymus is assessed. In one example,the agent increases T cell proliferation.

Thus, a transgenic mouse is disclosed whose somatic and germ cellscomprise a disrupted thymic stromal lymphopoietin receptor (TSLP) gene(the disruption being sufficient to inhibit the interaction of TSLP withits receptor). These mice show a decreased response to theadministration of an inflammatory antigen, such as an antigen known toinduce asthma. A transgenic mouse is disclosed whose somatic and germcells comprise a disrupted thymic stromal lymphopoietin receptor (TSLP)gene (the disruption being sufficient to inhibit the interaction of TSLPwith its receptor) and a disrupted γ_(c) gene (the disruption beingsufficient to reduce signaling through the γ_(c)). The disrupted IL-21receptor and disrupted γ_(c) genes are introduced into the mouse or anancestor of the mouse at an embryonic stage. A mouse homozyogous for thedisrupted TSLP receptor gene and homozygous for the disrupted γ_(c) genehas diminished thymic cellularity. A transgenic mouse is also disclosedwhose genome is heterozygous for an engineered disruption in a TSLPreceptor gene and whose genome is heterozygous for an engineereddisruption in an γ_(c) gene. The engineered TSLP receptor gene and theengineered γ_(c) gene in a homozygous state inhibits production of afunctional TSLP receptor and a functional γ_(c), such that thetransgenic mouse has reduced cellularity of the thymus as compared to awild-type mouse.

The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES Example 1 Materials and Methods

Generation of TSLPR^(−/−) mice: TSLPR genomic DNA was obtained from a P1clone prepared from 129sv mice (Genome Research). The targetingconstruct was designed to delete all exons of the TSLPR gene and replacethem with the neomycin resistance gene (Neo). The targeting constructhad 6 kb (Bgl II to Nhe I) and 3 kb (Pvu II) 5′ and 3′ flanking regions,respectively, of the Neo gene. Embryonic stem (ES) cells wereelectroporated with 50 μg of linearized targeting construct. Positiveand negative selection of transfected cells was conducted using G418(350 μg/ml; Life Technology, Carlsbad, Calif.) and gancyclovir (2 μM;Sigma, St. Louis, Mo.), respectively. Of 800 ES clones screened, thosethat had undergone homologous recombination were identified by Southernblotting, using probes located 5′ and 3′ to the targeting construct.Mice were mated once with wild-type C57BL/6. The mice were genotypedusing three PCR primers: TABLE 2 Genotyping Sequences Name SequenceIdentifier A 5′-AACCTCTCCCACAAGAAGTCCAGAAGT-3′ SEQ ID NO: 6 Neo5′-ATCGCCTTCTATCGCCTTCTT-3′ SEQ ID (N) NO: 7 B5′-AGACTTTACCTGATTCCTGCCTTG-3′ SEQ ID NO: 8

Primers A and B amplify a 250 bp segment of the TSLPR gene. Primers Nand B identify the targeted gene and give a 650 bp product. The probesfor Southern blots were generated using Taq Gold kit (AppliedBiosystems, Foster City, Calif.) under the following conditions: 94° C.for 2 minutes, 25 cycles of 94° C. for 30 seconds, 58° C. for 45 secondsand 45° C. for 45 second, then 72° C. for 7 minutes prior to cooling to4° C. Genotyping PCR reactions were performed under the same conditionsbut for 33 cycles instead of 25 and using “Ready to go PCR beads”(Amersham, Piscataway, N.J.). RT-PCR was used to determine TSLPR mRNAexpression. RNA samples were extracted from thymi using Trizol (Promega,Madison, Wis.) following the manufacturer's directions and 1 μg of totalRNA was used per reaction. Internal upstream(5′-GCGAGGGCGGGGCTGCTGGAG-3′, SEQ ID NO: 9) and downstream(5′-CCTGGCTGGCGGGGCTGTGGC-3′, SEQ ID NO: 10) primers were amplifiedusing RNA PCR kit (Applied Biosystems, Foster City, Calif.) according tomanufacturer's instructions, and Southern blots were performed usinggenomic DNA digested as described above. Probes were labeled,hybridized, and washed using QuickHyb protocol (Stratagene, La Jolla,Calif.) according to the manufacturer's recommendations. TSLPR^(−/−)mice generated from two different ES clones showed no apparentdifferences. Unless indicated otherwise, all mice analyzed were 6-8weeks of age littermates and generally sex-matched. No sex relateddifferences were observed between WT and TSLPR KO mice.

To generate TSLPR/γ_(c) double KO mice, TSLPR KO females were mated toγ_(c) KO males. As γ_(c) is on the X-chromosome, TSLPR^(+/−)γ_(c) ^(+/−)female and TSLPR^(+/−)γ_(c) ^(−/Y) male F1 progeny were then mated.TSLPR/γ_(c) double KO and γ_(c) KO littermate progeny were thenanalyzed.

Cytokine injections: Two week old WT or γ_(c) KO mice received dailyintraperitoneal injections with 0.1 ml of PBS alone or containing murineIL-7 (0.5 μg) or murine TSLP (0.5 μg) for 1 and 3 weeks, so that micewere 3 and 5 weeks old at the time of the analysis.

Treatment with IL-7 neutralizing mAb: Eight to ten week old WT and TSLPRKO females were irradiated with 600 cGy of whole body irradiation. Micewere injected 3 times a week for 4 weeks with 1 mg of a control mAb orM25 anti-IL-7 neutralizing mAb (Bhatia et al., J Exp Med 181:1399-1409,1995).

Flow cytometric analyses: Single cell suspensions were prepared fromthymus, spleen and bone marrow. Cells were washed with FACS buffer(phosphate buffered saline pH 7.4, 0.5% bovine serum albumin (BSA),0.02% sodium azide). One million cells were treated with Fc-block(PharMingen, San Diego, Calif.) for 15 minutes before being incubatedwith the indicated fluorochrome-conjugated antibodies (all fromPharMingen) for 20 minutes. Cells were then washed twice with FACSbuffer and analyzed.

Measurement of IgM levels: Sera from 3-week old γ_(c) KO and TSLPR/γ_(c)double KO mouse littermates were analyzed for resting IgM levels using asandwich enzyme linked immunosorbent assay (ELISA). Briefly, 100 μL (2μg/ml in coating buffer; 0.15 M sodium carbonate, 0.35 M sodiumbicarbonate pH 9.5, 0.02% sodium azide) of anti-mouse IgM captureantibody (PharMingen) was used to coat 96 well-plate (EIA plates,Costar, Somerset, N.J.) overnight at 4° C. Wells were coated withblocking buffer (PBS supplemented with 10% fetal bovine serum [FBS]) for1 hour at room temperature. Sera were diluted at 1:1000 in blockingbuffer and incubated overnight at 4° C. Wells were washed in PBScontaining 0.1% Tween and then incubated with a 1:2000 dilution ofsecondary HRP-conjugated anti-IgM (PharMingen) for 1 hour at RT. Theassay was ended by adding the substrate mixture (PharMingen) andmeasuring absorbance at 450 nm.

Proliferation and survival assays: To isolate CD4⁺ and CD8⁺ singlepositive T cells, thymocytes were first treated with anti-CD8⁺ oranti-CD4⁺ paramagnetic beads, respectively, and the cells that boundthese beads were removed by passing the samples through the autoMACSsystem (Miltenyi Biotec). The non-bound cells (negative eluted fraction)were then treated with CD4⁺ and CD8⁺ paramagnetic beads to separate CD4⁺and CD8⁺ cells, respectively, from the double negative cells. Maturesplenic CD4⁺ and CD8⁺ T cells were isolated using their respectiveparamagnetic beads. Fresh thymocytes and splenocytes were cultured for48 hours in RPMI 1640 medium containing 10% FBS, 2 mM L-glutamine, andantibiotics, on 96 well flat-bottom plates (2×10⁵ cells/well) that werenot coated or coated with anti-CD3ε (2 μg/ml, PharMingen). Cells wereadditionally incubated with IL-7 (100 ng/ml) or TSLP (100 ng/ml) asindicated. Wells were pulsed with 1 μCi of [³H] thymidine (6.7 Ci/mmol,NEN, Boston, Mass.) for the last 9 hours of culture. Proliferation wasalso examined using CFSE labeling (5 μM; Sigma, St. Louis, Mo.) for 10minutes at 37° C. For in vivo proliferation, 0.8 mg of5-Bromo-2′-deoxyuridine (BrdU) (Sigma-Aldrich, St. Louis, Mo.) wasinjected 16 and 10 hours before sacrifice. Levels of BrdU weredetermined using PE-conjugated antibodies (Tough et al., J Exp Med179:1127-1135, 1994). To examine the survival of thymocytes, cells werealso isolated and cultured as described above for 1 week and the numberof live cells was determined by trypan blue exclusion.

Adoptive transfer of T cells: γ_(c) KO mice were irradiated with 600 cGyof whole body irradiation. Eight hours later, the mice were injectedwith a single cell suspension of 8×10⁶ cells splenic CD4⁺ or CD8⁺ Tcells labeled with CFSE (10 minutes, 37° C.) that had been isolatedusing anti-CD4⁺ or anti-CD8⁺ paramagnetic beads (Miltenyi Biotec),respectively, from either WT or TSLPR KO mice. Recipient mice wereanalyzed on days 3 and 7 and host spleens were extracted and analyzed byflow cytometry.

Example 2 Generation of Mice Lacking TSLPR

In order to generate TSLPR KO mice, a targeting vector was used that wasdesigned to replace the TSLP coding region with the neomycin resistancecassette (FIG. 1 A). A total of 4 clones out of 800 showed homologousrecombination as determined using both 3′ and 5′ probes (FIG. 1 B). Twoclones were microinjected into blastocysts to generate chimeric mice andheterozygous offspring corresponding to each clone were intercrossed togenerate TSLPR^(−/−) mice, as confirmed by PCR genotyping, andevaluation of TSLPR mRNA expression (FIG. 1C). Breeding of TSLPR^(+/−)mice produced wild-type, heterozygous, and KO offspring in normalMendelian ratios. TSLPR^(−/−) mice were indistinguishable from wild-typelittermates in growth, development, breeding, and viability. Theinactivation of TSLP signaling was confirmed by the lack of a greaterresponse of TSLPR KO splenocytes to treatment with anti-CD3+TSLP thananti-CD3 alone, as opposed to the greater response seen in wild-type(WT) splenocytes when anti-CD3 and TSLP were combined (FIG. 1D).

Example 3 TSLPR KO Mice Exhibit Normal Lympho-Hematopoietic Development

Thymus, spleen, and bone marrow were similar in size and cell number,and no differences in histology were observed in TSLPR KO mice versustheir wild-type littermates. Analysis of TSLPR KO thymi showed that thedouble negative (DN), double positive (DP), and CD4⁺ and CD8⁺ singlepositive T cells were present at normal distributions (FIG. 1E).Furthermore, mature T cells in the spleen were also present at theexpected ratios.

B220 versus anti-CD3 staining in the spleen was normal, as was theCD4:CD8 ratio T cell ratio (FIG. 1 F, upper panels). Moreover, levels ofboth B220⁺ IgM⁺ immature cells and B220⁺ IgM⁻ pre-B and pro-B cells(both percent and absolute numbers) were similar in TSLPR KO and WT micein both spleen (FIG. 1 F, lower panels) and bone marrow (FIG. 1 G).Surface levels of CD3, TCR β, TCR γδ, CD4, CD5, CD8, CD11c, CD21, CD23,CD43, CD44, CD62L, B220, IgM, IgD, DX5, Gr1, TER119, Yc, HSA, and BP-1,as well as IL-7Rα, were comparable to those found in wild-type mice.Thus, although TSLP has been reported to affect fetal lymphoiddevelopment, no overt defects were found in adult lymphopoiesis in TSLPRKO mice. It is possible that TSLP serves a redundant role and that othercytokines, especially the closely related IL-7, could compensate for theabsence of TSLP signal. No difference was observed between WT and TSLPRKO mice in the number of colony forming units (CFU-B and CFU-GM assays)from BM cultures in vitro. Although TSLPR is expressed in varioustissues including brain, liver, and lung (Pandey et al., Nat Immunol1:59-64, 2000), suggesting a possible role of TSLP in the physiologicalfunctions of these organs, histopathological analysis of these organs inTSLPR KO mice revealed no obvious abnormalities.

Example 4 TSLP and IL-7 Exhibit Overlapping Actions In Vivo

Because TSLP and IL-7 share IL-7Rα as a receptor component, the actionsof these cytokines was compared. Both IL-7 and TSLP similarly increasedthymic and splenic cellularity in WT mice after one week of dailyinjections (FIG. 2 A, left half of panel), with CD4 and CD8 T cellpopulations present in normal ratios (FIG. 9A, ii and iii versus i; vand vi versus iv); thus, thymic and splenic T cell populations were allincreased. FIG. 9 shows the effect of TSLP on lymphopoiesis in WT mice.Shown are the flow cytometric analysis of thymus and spleen 1 and 3weeks after injection of WT mice with PBS, IL-7, or TSLP. Thymocyteswere stained with anti-CD4 versus anti-CD8; splenocytes were stainedwith anti-CD4 versus anti-CD8 and anti-B220 versus anti-IgM.

In the spleen, TSLP and IL-7 had similar abilities to increase B cells(FIG. 9A, viii and ix versus vii), while TSLP was more potent inexpanding immature B220⁺ IgM^(low) cells in the BM (FIG. 2B, ii and iiiversus i; see percentages for the different fractions of B cells in FIG.2C). After three weeks of treatment of wild-type mice with TSLP or IL-7,the increase in thymic and splenic cellularity was no longerstatistically significant (FIG. 2A, right half of panel), and theCD4⁺/CD8⁺ T cell ratio remained constant (FIG. 9B, ii and iii versus iand v and vi versus iv), but the increase in B cells was still evidentin the spleen (FIG. 9B, viii and ix versus vii) and BM (FIG. 2B, v andvi versus iv; see percentages for the different fractions of B cells inFIG. 2C). The consistent absolute decrease in B cells in WT animalsbetween 1 and 3 weeks of treatment presumably at least in part reflectsan age-dependent regulatory mechanism of B cell expansion, as a decreasewas even seen in the untreated control animals (FIG. 2 C). These resultsindicate that both TSLP and IL-7 can promote lymphocyte expansion. Thenormal levels of lymphocytes in TSLPR KO mice presumably are likely inpart or entirely explained by the continued action of IL-7.

Example 5 TSLP Signaling is Required for Efficient Recovery ofLymphocyte Populations Following Sub-Lethal Irradiation

To further examine the role of TSLP in lymphopoiesis, WT and TSLPR KOmice were sub-lethally irradiated and their ability to recover cellularpopulations was evaluated. In these experiments, either a control mAb orneutralizing mAb to IL-7 was included. Irradiated WT animals treatedwith the control mAb recovered most of their thymic and spleniccellularites within 4 weeks, whereas TSLPR KO mice did not (FIG. 3 A).Similarly, CD4⁺ and CD8⁺ T cell sub-populations in the thymus (FIG. 3 B)and spleen (FIG. 3 C) were lower in TSLPR KO mice than in WTlittermates. B cell recovery in TSLPR KO mice was also less efficientthan in WT mice (FIG. 3C), and flow cytometric analysis of B cellsshowed that B220⁺ CD19⁺ B cells were greatly diminished in the spleensin TSLPR KO mice (FIG. 3D). These results establish a critical role forTSLP in mediating optimal T and B cell lymphopoiesis in mice.

Reconstitution was examined in mice in which a neutralizing mAb to IL-7was injected (1 mg three times a week for 4 weeks) (Bhatia et al., J ExpMed 181:1399-1409, 1995). As expected, treatment with the anti-IL-7 mAbreduced lymphopoiesis in WT mice (FIG. 3A). Importantly, however,recovery was even more impaired in TSLPR KO mice treated with anti-IL-7mAb, including impaired thymic and splenic cellularity (FIG. 3A), withCD4⁺ and CD8⁺ T cell sub-populations in the thymus (FIG. 3 B) and spleen(FIG. 3C) being lower in TSLPR KO mice than in WT littermates. Nosignificant differences in the percentages of DN1, DN2, DN3, and DN4cells were observed. B cell recovery in TSLPR KO mice was also lessefficient than in WT mice in the presence of anti-IL-7 mAb (see bonemarrow and spleen in FIGS. 3C and 3D). B220⁺ CD19³⁰ B cells were nearlyeliminated in the BM in both WT and TSLPR KO mice treated with anti-IL-7(FIG. 3D, far right panels). These results establish a critical role forTSLP in mediating optimal T and B cell lymphopoiesis in mice. Becauserecovery is less in the absence of signaling by TSLP+IL-7 as compared tothe absence of only IL-7, the data presented herein indicate a criticalIL-7-independent role for TSLP in recovering from lymphopenia.

Example 6 TSLPR/γ_(c) Double KO Mice Exhibit Greater Defects than doγ_(c) KO Mice

γ_(c) KO mice have defective T and B cell development. To furtherinvestigate a possible distinctive role of TSLP in lymphoid development,TSLPR KO mice were crossed to γ_(c) KO mice to generate F2 progenylacking both receptor chains (see Methods). γ_(c) KO mice have defectiveT and B cell development. A greater defect in γ_(c)/TSLPR double KO micethan in γ_(c) single KO mice would suggest a role for TSLP incontributing to the residual lymphoid development that is present inγ_(c) deficient mice and in humans with XSCID. Indeed, the low thymiccellularity observed in γ_(c) KO mice (DiSanto et al., Proc Natl AcadSci USA 92:377-381, 1995; Cao et al., Immunity 2:223-238, 1995) wasfurther diminished in double KO littermates (FIG. 4A). However, CD4⁺ andCD8⁺ single positive, CD4⁺CD8+double positive (DP), and CD4⁻CD8⁻ doublenegative T cells (DN1, DN2, DN3 and DN4) were all present at ratioscomparable to thymi of γ_(c) deficient mice. Similarly, although totalBM cellularity was greatly reduced, a similar distribution of B cellsub-populations was present in BM (FIG. 4B) and spleen as were seen inγ_(c) KO mice. Specifically, B220 versus CD43 profiles were similar inboth γ_(c) KO and the γ_(c)/TSLPR double KO mice (FIG. 4B, upperpanels), as was expression of HSA, BP 1, and IgM (data not shown).Peritoneal CD5⁺ B1 cells were also present in similar numbers in bothγ_(c) KO and the γ_(c)/TSLPR double KO mice (FIG. 4B, lower panels).Consistent with this, γ_(c)/TSLPR double KO mice did not have lowerserum IgM levels than those found in γ_(c) KO mice (FIG. 4C). Thus, TSLPdoes not appear to regulate numbers of B1 cells nor affect IgMproduction.

Example 7 TSLP Expands Both B and T Cells in γ_(c) KO Mice

As an alternative approach to elucidate a possible role for TSLP inlymphoid physiology, the effect of TSLP on γ_(c) KO mice was evaluated.There is a sharing of IL-7Rα by the receptors for both of thesecytokines and the potential for overlapping actions between TSLP andIL-7. It was possible that these γ_(c) KO mice that lack IL-7 signalingand have diminished thymocytes could exhibit hyper-responsiveness toTSLP. Two week old γ_(c) KO mice received daily 0.5 μg i.p. injectionsof phosphate buffered saline (PBS) or TSLP for 1 or 3 weeks. Strikingly,after 1 week, TSLP treatment increased thymic cellularity 5-10 fold(p<0.01) (FIG. 4D, open rectangles versus ovals), consistent withincreased thymic size (FIG. 4E, upper panels). Histological analysisshowed that thymi from γ_(c) KO mice injected with PBS had a thin cortexwhereas the thymi of TSLP-injected mice had a wider corte (FIG. 4E.lower panels).

In addition to increased thymic cellularity, the fraction of DP cellsincreased so that this was the most expanded thymic population (FIG. 5A,ii versus i). γ_(c) KO mice injected with TSLP for 3 weeks still showedhigher total thymic cellularity than untreated mice (FIG. 4D, p<0.01)albeit less so than at 1 week, with more CD4⁺ SP T cells present (FIG.5A, x versus ix). TSLP treatment for one week also increased the totalnumber of splenocytes (p<0.01) (FIG. 4D), mainly by increasing B cells(FIG. 5A, vi versus v). After three weeks of TSLP, splenic cellularityfurther increased (p<0.01) (FIG. 4 D). As expected, this resulted inpart from the expansion of B cells (FIG. 5 A, xiv versus xiii). Theincrease in B cells in the spleen was also evident in the bone marrow.Although the total BM cellularity in γ_(c) KO mice was not increased byTSLP, TSLP dramatically increased the B cell sub-populations within 1week (FIG. 5A, viii versus vii), with immature B cells being the mostaffected, increasing from approximately 2% to 30% of the total number ofcells (see % of B220⁺ IgM^(low) cells in FIG. 5B). More mature stagesalso increased three to four fold. However, these changes in B cellpopulations were transient, and at three weeks of TSLP injection thesecells diminished to the BM B lymphocyte cellularity seen in theuntreated mice (FIG. 5A, xvi versus xv and FIG. 5B).

In addition to the increased B cells noted above, the increased spleniccellularity was also partially due to an increase in CD4⁺ T cells (FIG.5A, xii versus xi and FIG. 5C). Consistent with the age-dependentincrease in CD4⁺ T cells that occurs in γ_(c) KO mice (Cao et al.,Immunity 2:223-238, 1995), control γ_(c) KO mice that were injected withPBS for 3 weeks versus one week showed an age-dependent increase in thepercentage of CD4⁺ T cells (FIG. 5A, xi versus iii). Approximately 5.4fold more CD4⁺ splenic T cells were found in TSLP-treated γ_(c) KO micethan in untreated mice (12.7×10⁶ versus 2.4×10⁶), whereas the absoluteand relative increase in mature splenic CD8⁺ T cell numbers was lessmarked in TSLP-treated versus control animals (3.8 fold, 2.4×10⁶ versus0.63×10⁶) (FIG. 5C). Almost all of the CD4⁺ T cells in γ_(c) KO micewere TCR^(low) HSA^(low) and did not express the activation markers CD25and CD69 and this was unaffected by TSLP treatment (FIG. 5D, upper andmiddle panels). CD4⁺ T cells in γ_(c) KO mice primarily have aCD62^(low) CD44^(high) memory phenotype (Nakajima et al., J Exp Med185:189-195, 1997). Interestingly, TSLP most increased the number ofCD62L^(high) CD44^(high) cells (FIG. 5D, lower panels), a populationthat has been identified as central memory cells (Sprent and Surh, CurrOpin Immunol 13:248-254, 2001).

Example 8 TSLP Induces Thymocyte Proliferation and Cooperates withAnti-CD3 to Preferentially Expand CD4⁺ T Cells

Thus, an increase in the number of CD4⁺ T cells was observed,particularly in the spleen but also in the thymus. To further examinethe effect of TSLP on T cells, the effect of TSLP injection on the invivo proliferation of thymocytes was examined. γ_(c) KO mice treatedwith PBS or TSLP for 1 week were injected with BrdU 10 or 16 hoursbefore sacrifice. TSLP increased BrdU incorporation in allsubpopulations of thymocytes (FIG. 5E). Interestingly, γ_(c) KO CD4⁺ SPthymocytes had the lowest basal level of BrdU incorporation and were themost responsive to TSLP treatment, increasing their proliferationapproximately four fold. Thus, TSLP enhanced the proliferation of allthymocyte subpopulations.

To clarify the mechanism by which TSLP promotes thymocyte expansion,single positive WT thymocytes were treated in vitro with anti-CD3ε withor without IL-7 or TSLP. TSLP alone had no significant effect on theproliferation of either CD4⁺ or CD8⁺ SP thymocytes. However, whencombined with anti-CD3ε, TSLP markedly increased the proliferation ofCD4⁺ SP thymocytes while its effect on CD8⁺ SP thymocytes was much less(FIG. 6A, upper panel), suggesting that TSLP favors the expansion ofCD4⁺ thymocytes. In contrast, IL-7 enhanced proliferation of both CD4⁺and CD8⁺ SP thymocytes alone or in combination with anti-CD3ε (FIG. 6A,lower panel), and overall, IL-7 was more potent then TSLP even for CD4⁺cells (note the difference in scale in the upper and lower panels ofFIG. 6A). The ability of TSLP to influence the survival of SP thymocyteswas also examined in vitro. Correspondingly, TSLP preferentiallyenhanced the survival of CD4⁺ thymocytes, whereas IL-7 had similareffects on viability of CD4⁺ and CD8⁺ thymocytes (FIG. 7A). TSLP andIL-7 were equally potent in increasing the survival of DN and DPthymocytes.

Consistent with its effect on CD4⁺ SP thymocytes, TSLP treatmentpreferentially enhanced TCR-induced proliferation (FIG. 6B) and survival(FIG. 7B and FIG. 7C) of CD4⁺ splenic T cells as compared to CD8⁺ Tcells. Examining the proliferation of thymic and splenic CD4⁺ cellsusing CFSE staining showed a dramatic dilution of CFSE staining inresponse to TSLP in both (FIG. 6C). Correspondingly, the percent ofviable cells was increased (FIGS. 7A and 7B) and the percent of AnnexinV⁺/7-AAD⁺ cells (FIG. 7 C) was decreased by TSLP.

As compared to CD4⁺ T cells from WT mice, CD4⁺ T cells from TSLPR KOmice exhibited less expansion during the 7 day period after injectioninto irradiated γ_(c) KO mice (FIG. 8A), indicating that TSLP signalingis critical for an efficient expansion of CD4⁺ lymphocytes even in anenvironment where IL-7 is not limiting and where endogenous CD4 cellsexpansion could occur. CFSE staining on day 3 showed the typical four tofive divisions in WT CD4⁺ T cells (FIG. 8B, upper left panel); however,TSLPR KO CD4⁺ T cells persisted but had undergone fewer cell divisions(FIG. 8B, upper right panel). In contrast, CD8⁺ T cells from WT andTSLPR KO mice were similar in their CFSE profiles. Taken together, theseresults indicate that TSLP preferentially enhances both the expansionand survival of CD4⁺ T cells both in vitro and in vivo.

TSLP was discovered as a cytokine that could support the ability ofpre-B cells to differentiate into more mature IgM⁺ B cells, whereas IL-7promotes development only to an IgM⁻ stage (Friend et al., Exp Hematol22:321-328, 1994). Colonies emerging from murine B220⁺ IgM⁻ bone marrowcells develop into IgM⁺ cells after 7 days of culture in TSLP. Incontrast, TSLP was previously shown to play only a minimal role inmurine T-lymphopoiesis by inducing proliferation of double negativethymocytes, but only in an IL-1-dependent manner (Sims et al., J Exp Med192:671-680, 2000).

To clarify TSLP function, TSLPR deficient mice have been studied theeffects of TSLP have been examined in vivo and in vitro. Despiteubiquitous expression, TSLPR is not required for the physicaldevelopment and fertility of mice. In addition, TSLPR KO mice exhibitnormal myeloid, lymphoid, dendritic cell and NK cell numbers, at leastin part due to the continuous action of IL-7. To examine the role ofTSLP in lymphopoiesis, TSLPR KO mice were crossed to γ_(c) KO mice.Inactivating TSLP signaling in γ_(c) KO mice further reduced thecellularity of the thymus, spleen, and BM, suggesting that TSLP canpromote T and B cell expansion. Nevertheless, the existence oflymphocytes in these double KO mice indicates that other growth factorsalso contribute.

Cellular recovery following sub-lethal irradiation of TSLPR versus WTmice was evaluated. The defective cellular restoration in TSLPR KO micesuggests that TSLP is of use recovery from lymphopenia. Moreover, thedefect was also more severe in TSLPR than WT mice injected withneutralizing antibodies to IL-7, indicating that TSLP has at least someactions that are independent of IL-7. Interestingly, however, inJak3/TSLPR double KO mice, a decrease was observed in B cells in thespleen but not in the BM and saw no change in T cells. In contrast, inthe above-described analysis of γ_(c)/TSLPR double KO mice, totalcellularity declined with marked decreases in both B and T cells,findings that are consistent with a role for TSLP in γ_(c)-independentexpansion of both of these lineages.

TSLP promoted B cell maturation in γ_(c) mice to the B220⁺IgM⁺ stage. Italso enhanced B-cell maturation in WT mice to an almost identical levelas IL-7, similar to what was reported in neonatal wild-type miceinjected with TSLP (Sims et al, J Exp Med 192:671-680, 2000).Interestingly, B progenitor cells in the BM of γ_(c) KO mice expandedand matured when animals were injected daily with TSLP for 1 week, butthe effect was transient and no longer observed after three weeks ofTSLP. TSLP had less of an effect in WT mice. Our findings in γ_(c) KOmice could also reflect changing potentials of the progenitor populationin these young animals.

A distinctive action of IL-7 is its ability to increase survival ofimmature thymocytes and provide a proliferative signal in the pre-T cellstage after TCR-β rearrangement (Zlotnik et al., Curr Opin Immunol7:206-213, 1995). IL-7 is also essential for γδ TCR generation (Moore etal., J Immunol 157:2366-2373, 1996). Although TSLP could not rescue γδ Tcell development when it was injected into γ_(c) KO mice, like IL-7,TSLP could promote the survival and proliferation of T cells as well asin B cells from WT mice. Strikingly, TSLP can preferentially increaseTCR-mediated proliferation of CD4⁺CD8⁻ thymocytes and CD4⁺ peripheral Tcells in vitro (FIG. 6), and the absence of TSLP signaling hinders theexpansion of these cells in vivo in the adoptive transfer experiment(FIG. 8). The modest effect of TSLP on CD4⁺ T cell expansion seen inwild-type mice (FIG. 2) could be related to the presence of IL-7, whichin addition to its effect on CD8⁺ T cells homeostasis, can also promoteCD4⁺ T cell survival (Seddon et al., Nat Immunol 4:680-686, 2003).Without being bound by theory, it is also possible that the T cellcompartment is already “filled”, allowing less expansion and/or that itis less responsive as cells have already received signals fromγ_(c)-dependent cytokines so that a potentially redundant signal fromTSLP would have little effect. Interestingly, two other γ_(c) cytokines,IL-7 and IL-15, preferentially induce an expansion of CD8⁺ T cellsrather than CD4⁺ T cells (Kieper et al., J Exp Med 195:1533-1539, 2002;Schluns et al., Nat Rev Immunol 3:269-279, 2003; Lodolce et al.,Immunity 9:669-676, 1998).

Like IL-7 and IL-15, TSLP activates Stat5a and Stat5b (Isaksen et al., JImmunol 163:5971-5977, 1999; Isaksen et al., J Immunol 168:3288-3294,2002). Stat5a and Stat5b transgenic mice show an increase in CD8⁺ Tcells (Kelly et al., J Exp Med 198:79-89, 2003; Kelly et al., J Immunol170:210-217, 2003) which suggests that Stat5 by itself is unlikely to bemediating the TSLP effect on CD4⁺ T cell expansion. Unlike IL-7, TSLPhad no effect on the in vitro proliferation of thymocytes unlesscombined with TCR activation. Since the differentiation of CD4⁺CD8⁺thymocytes into single positive cells is based on MHC-specificity oftheir TCR signal and the strength of the TCR engagement (Germain et al.,Nat Rev Immunol 2:309-322, 2002), it is conceivable that TSLP provides aselective co-stimulatory signal that favors the CD4⁺ CD8⁻ intermediatestage by enhancing the activation of these cells. Moreover, TSLP couldcompete with IL-7 for the IL-7Rα subunit, thus hindering the ability ofIL-7 to promote CD8⁺ T cell expansion in WT mice. Interestingly, IL-7has been suggested to be important as a survival factor for CD4 memorycells (Seddon et al., Nat Immunol 4:680-686, 2003). The resultsdescribed above in the murine system with purified CD4⁺ and CD8⁺ SPthymocytes as well as splenic T cells suggest a direct effect of TSLP onCD4⁺ T cells. Thus, TSLP and IL-7, both of which share IL-7Rα, likelyare important for CD4⁺ T cell homeostasis.

Thus, evidence is provided herein that demonstrates that TSLP plays arole in T cell expansion both in vitro and in vivo, particularly of CD4⁺T cells. TSLP could play a role in CD4⁺ T cell homeostasis. Thispreferential action of TSLP for CD4⁺ versus CD8⁺ T cells likely explainsthe relative augmentation in CD4⁺ T cells in γ_(c) KO mice.

Example 9 Materials and Methods for Example 10

Mice: TSLPR KO mice are described above. DO11.10 transgenic Rag 2^(−/−)mice on B10.D2 and Balb/c backgrounds (purchased from Taconic) werecrossed to TSLPR KO mice on F1 129/BL/6 or Balb/c (F4) geneticbackgrounds. WT Balb/c animals were from the Jackson Laboratory. TSLPeffect on naïve versus memory CD4 T cells: CD4⁺CD62L⁺CD44^(low) (naïve),CD4⁺CD62L⁺CD44^(high) (central memory), and CD4⁺CD62L⁻CD44^(high)(effector memory) phenotype T cells were isolated by cell sorting andwere >99% pure. RNA was extracted from freshly sorted cells using Trizol(Invitrogen). Purified cells were also suspended at (2×10⁵ cells/well)and activated with anti-CD3ε (2 μg/ml, Pharmingen) with or without TSLP(100 ng/ml, R and D) for 48 hours before being pulsed with 1 μCi of [³H]thymidine (6.7 Ci/mmol, NEN, Boston, Mass.) for the final 16 hours ofculture.

Immunization and antigen-recall response: Mice were immunized byintra-peritoneal injection of 200 μg of ovalbumin (OVA) (Pierce,Rockford, Ill.) in 100 μl of PBS that had been emulsified with an equalvolume of Aluminum Hydroxide (ALUM) as adjuvant. Mice were sacrificed 12or 60 days after immunization. For assessing an antigen recall response,purified CD4⁺ T cells (1×10⁵ cells/well) were cultured with an equalnumber of splenic antigen presenting cells (APCs) or total splenocytes(2×10⁵ cells/well) that were treated with mitomycin C (50 μg/ml in PBSfor 15 minutes at 37° C.) and washed 3 times. These cells were thenincubated in 96 well flat-bottom plates for 48 hours in RPMI 1640 mediumcontaining 10% FBS, 2 mM L-glutamine, and antibiotics, and with 0, 10,50 or 200 μg/ml of OVA. Wells were pulsed with 1 μCi of [³H] thymidine(6.7 Ci/mmol, NEN, Boston, Mass.) for the final 16 hours of culture.Where indicated, APCs included B cells, macrophages, and dendritic cells(DC), after depleting CD4⁺ T cells, CD8⁺ T cells, and natural killer(NK) cells by positive selection. Wild-type (WT) and TSLPR KO miceexpressing the DO11.10 transgene were treated with 100 μg of OVAemulsified in ALUM and analyzed the next day.

Dendritic cell function: TSLP was from R&D Biosystems and testednegative for endotoxin by the LAL (Limulus Amebocyte Lysate) test(BioWhittaker, Md.). To examine the effect of TSLP on murine DC, totalsplenic CD11c⁺ DC were isolated by sorting (>99% purity) from Balb/c WTmice and cultured for 24 hours at 1×10⁶ cells/ml with OVA³²³⁻³³⁹ peptide(5 μg/ml) (Bachem, King of Prussia, Pa.) with or without TSLP (100ng/ml). The cells were lysed in Trizol for RNA isolation or were washed,treated with mitomycin C (50 μg/ml, Sigma), washed three times, andincubated with DO11.10 RAG 2^(−/−) CD4⁺ T cells at 1:10 ratio. Cellswere either cultured for 48 hours to allow measuring proliferation using[³H] thymidine incorporation or for four days to examine intracellularlevels of IFN-γ and IL-4 secretion, as described below.

“Mix and match” experiments were performed using CD4⁺ T cells purifiedby positive selection using specific magnetic beads (Miltenyi Biotec).CD4⁺ T cells and CD11c⁺ DC were isolated by positive selection usinglabeled magnetic beads (Miltenyi Biotec) in the presence of 1% Fc block(PharMingen). The resulting CD4⁺ T cells were >90% pure and DC were >80%pure. They were incubated together (1:10 ratio) at various combinations(for example, WT CD4⁺ T cells with WT or TSLPR KO DC and TSLPR KO CD4⁺ Tcells with WT or TSLPR KO DC) as indicated. All DC were treated withmitomycin C before being incubated with CD4⁺ T cells. Wells were pulsedwith 1 μCi of [³H] thymidine (6.7 Ci/mmol, NEN, Boston, Mass.) for thefinal 16 hours before harvesting.

Flow cytometric analyses: Single cell suspensions were prepared fromthymus and spleen. Cells were washed with FACS buffer (phosphatebuffered saline pH 7.4 containing 0.5% bovine serum albumin (BSA) and0.02% sodium azide). One million cells were treated with Fc-block for 15minutes before being incubated with the indicatedfluorochrome-conjugated antibodies (all from PharMingen) for 20 minutes.Cells were then washed twice with FACS buffer and analyzed. Allantibodies used were from PharMingen except KJ1-26 mAb, which was fromCaltag Laboratories (Burlingame, Calif.).

Culture under polarizing conditions: Purified CD4⁺ T cells wereactivated with anti-CD3 (2 μg/ml) and anti-CD28 (1 μg/ml) and culturedin conditions favoring Th1 (1 ng/ml IL-12 and 10 μg/ml anti-IL-4) or Th2(1 ng/ml IL-4 and 20 μg/ml anti-IFN-γ) in the presence or absence ofTSLP (100 ng/ml). IL-2 (100 U/ml) was added on day 2 and cells wereallowed to expand for 1 week.

Intracellular staining for IFNγ and IL-4 levels: Cultured cells wereactivated with PMA (10 ng/ml) and ionomycin (1 μg/ml) (both from Sigma)for 5 hours in the presence of Golgi Block (PharMingen). Cells werewashed and stained for either anti-CD4 (PharMingen) and/or for theDO11.10 specific transgene with KJ1-26. Intracellular staining wasperformed using Cytofix/Cytoperm kit and anti-IFN-γ and IL-4 (all fromPharMingen) according to the manufacturer's instructions.

Sensitization and airway challenge to mice: OVA (100 μg) was emulsifiedat a 1:1 ratio in ALUM in 200 μl and injected intraperitoneally on days0 and 7 to sensitize mice. On day 13, mice were challengedintratracheally with 50 μg of OVA in 30 μl PBS. On day 14, 50 μg of OVAin 25 μl PBS was administered intranasally. Control mice were treatedsimilarly with PBS instead of OVA. On day 16, mice were sacrificed,bled, and bronchoalveolar lavage (BAL) was performed using 0.5 ml ofPBS. Cells from BAL fluid were isolated by cytospin and leukocytesidentified by staining using Wright/Giemsa. Lungs were either used forextracting RNA or for generating tissue sections for microscopicanalysis. Lung tissue sections were stained with either Periodic-AcidSchiff (PAS) or Wright/Giemsa (Volu-Sol, Utah). Serum levels ofOVA-specific immunoglobulin were measured by ELISA (PharMingen).Inflammation was scored on a scale of 0 to 4 as follows: (0) Normallungs, no goblet cell hyperplasia; (1) Minor perivascular inflammationaround large blood vessels; (2) Moderate perivascular and peribronchialinflammation, minimal evidence of goblet cell hyperplasia; (3) Increasedperivascular and peribronchial inflammation with increased goblet cellhyperplasia beginning in smaller airways; (4) Severe formation ofperivascular, peribronchial, and interstitial inflammation as well asgoblet cell hyperplasia in both small and large airways.

Adoptive transfer of CD4⁺ T cells: WT and TSLPR KO mice backcrossed toBalb/c background for four generations were designated as donors andrecipients and immunized by intraperitoneal injections of OVA (100 μg)emulsified at a 1:1 ratio in ALUM in 200 μl on days 0 and 7. On day 13,recipient mice were sensitized intratracheally with 50 μg of OVA in 30μl PBS, followed 4 hours later by i.v. transfer of 8×10⁶ CD4⁺ T cellsextracted from spleen and lymph nodes of donor mice. On day 14, 50 μg ofOVA in 25 μl PBS was administered intranasally. Mice were sacrificed andanalyzed the next day for signs of lung inflammation, using the criteriapresented above.

Real time PCR: RNA was extracted from single cell suspensions or fromlung tissue using Trizol (Invitrogen). RNA was reverse transcribed usingthe RNA PCR gold kit (Applied Biosystems). Levels of IL-2, IL-4, IL-5,IL-10, IL-13, IFN-γ, TSLP, and TSLPR mRNA relative to 18S rRNA weremeasured by RT-PCR using “Gene expression assays” ready-made primersfrom Applied Biosystems.

Example 10 Further Analysis of the Effect of TSLP

To better understand the function of TSLP in immune responses and toclarify the CD4⁺ T cell population(s) on which it acts, the effect onnaïve (CD62L⁺ CD44^(low)), central memory (CD62L⁺ CD44^(hi)) andeffector memory (CD62L⁻ CD44⁺) CD4⁺ T cell populations was examined.TSLP significantly enhanced the anti-CD3 induced proliferation of naïveCD4⁺ T cells from Balb/c mice, but had little effect on the expansion ofmemory phenotype CD4⁺ T cells (FIG. 10A).

To further analyze the effect of TSLP in vivo, TSLPR KO mice werecrossed to DO11.10 transgenic (Tg) mice and analyzed mice that expressedthe TCR transgene and either expressed (DO11.10/WT) or lacked(DO11.10/TSLPR KO) the TSLPR gene. Splenic CD4⁺ T cells from theseanimals were examined; DO11.10/WT and DO11.10/TSLPR KO CD4⁺ T cellsexpressed little if any CD69, an early T-cell activation marker (FIG.10B, upper panels). However, injection of mice with ovalbumin (OVA)significantly induced CD69 on DO11.10/WT CD4⁺ T cells but notDO11.10/TSLPR KO (FIG. 10B, lower panels), indicating that the absenceof TSLP signaling diminishes the activation of naïve CD4⁺ T cells byantigen in vivo.

The role of TSLP in TCR-driven generation of memory T cells from naiveCD4⁺ T cells was examined. WT and TSLPR KO mice on a Balb/c WTbackground (that do not express the DO11.10 transgene) were immunizedwith OVA. As evaluated by an in vitro antigen recall assay, splenocytesisolated from TSLPR KO mice 12 or 60 days after immunization had a lowerproliferative response to secondary exposure to the antigen than didsplenocytes from WT mice (FIGS. 10C and 10D). CD4⁺ T cells were isolated60 days after immunization and cultured with antigen presenting cells(APC) at a 1:1 ratio in the presence of OVA. CD4⁺ T cells from TSLPR KOmice showed much weaker proliferation in response to secondary antigenexposure than did cells from WT littermates (FIG. 10E). These resultssuggest that TSLP is primarily involved in antigen-driven activation ofnaïve CD4⁺ T cells and its absence significantly diminishes thegeneration of memory T cells.

Example 11 TSLPR Plays a Role in the Activation of Dendritic Cells inMice

The effect of TSLP on the contribution of APCs to the expansion of CD4⁺T cells in response to secondary stimulation was examined in vitro. WTand TSLPR KO mice were immunized with OVA, sacrificed at day 60, andsplenic CD4⁺ T cells were purified and incubated at a 1:1 ratio withAPCs in the presence of OVA. WT CD4⁺ T cells cultured with WT APCsproliferated the most. Replacement of WT APCs with TSLPR KO APC mildly(but significantly) reduced T-cell proliferation (FIG. 11A). TSLPR KOCD4⁺ T cells showed the weakest proliferative response to antigen, andthis was not significantly increased by introducing WT APC (FIG. 11A).Thus, TSLP has an essential role for CD4⁺ T cells, but the decreasedproliferation of WT CD4⁺ T cells when TSLPR KO APC were used suggeststhat TSLP signaling is required for the optimal activity of APCs aswell.

In view of the findings above, the role of murine TSLP on WT DC wasinvestigated. It was confirmed that WT DC express TSLPR mRNA usingRT-PCR. When WT splenic CD11c⁺ DC were activated with OVA³²³⁻³³⁹ peptidefor 24 hours, the addition of TSLP moderately increased the cell surfaceexpression of CD80, CD86, and MHC II (FIG. 11B). As compared tountreated DC, TSLP-treated DC also significantly enhancedpeptide-mediated proliferation of DO11.10 TCR transgenic CD4⁺ T cellswhen the DC and T cells were incubated in a 1:10 ratio (FIG. 11C).

To further examine the effect of TSLP on the ability of DC to activatenaïve cells, splenic CD4⁺ T cells and DC were isolated fromnon-immunized DO11.10/WT and DO11.10/TSLPR KO mice, and cultured DC withCD4⁺ T cells at a 1:10 ratio in the presence of 5 μg/ml of OVA³²³⁻³³⁹peptide. As expected, DO11.10/WT CD4⁺ T cells cultured with WT DC showedthe highest levels of proliferation. This was slightly but significantlyreduced when DO11.10/WT CD4⁺ T cells and DO11.10/TSLPR KO DC were used(FIG. 11D). The weak expansion seen when TSLPR KO CD4⁺ T cells wereincubated with TSLPR KO DC was not significantly enhanced when WT DCwere used. These results indicate that TSLP activates murine DC, butthat the presence of TSLPR on CD4⁺ T cells is more critical than itspresence on DC for optimal proliferation to the OVA³²³⁻³³⁹ peptide,consistent with the results shown in FIG. 11A.

Example 12 TSLP-Treated DC Reduce IFN-γ Production by CD4⁺ T Cells

Because TSLPR is critical for naive CD4⁺ T-cell proliferation, it wasnext examined whether TSLP could influence the cytokines produced byCD4⁺ T cells in response to TCR stimulation. Naive CD4⁺ CD62L⁺CD44^(low)and effector memory CD4⁺ CD62L⁻CD44^(hi) splenic T cells were purifiedfrom WT Balb/c mice and activated the cells with anti-CD3 in thepresence or absence of TSLP. On day 4, cells were stimulated withPMA+ionomycin for 5 hours and intracellular levels of IFN-γ and IL-4were determined. TSLP did not affect the level of IFN-γ or IL-4 in naiveor memory CD4⁺ T cells under neutral conditions (FIG. 12A). Furthermore,when cells were activated using Th1 or Th2 polarizing conditions, theaddition of TSLP did not affect the levels of either cytokine (FIG.12B).

It was next examined whether TSLP could indirectly influence thedifferentiation of CD4⁺ T cells by its action on DC. Sorted WT splenicCD11c⁺ DC were pre-incubated with OVA³²³⁻³³⁹ in the presence or absenceof TSLP before being washed and incubated with congenic DO11.10 Tg CD4⁺T cells (on a RAG2^(−/−) background). Treatment of DC withTSLP+OVA³²³⁻³³⁹ reduced the levels of IFN-γ production in KJ1-26⁺CD4⁺DO11.10 Tg T cells (FIG. 12C). Under these conditions, the levels ofIL-12 produced by DC were below the level of detection sensitivity byELISA. Real time PCR showed treatment of DC with OVA³²³⁻³³⁹+TSLP versusOVA³²³⁻³³⁹ alone tended to decrease IL-12 mRNA levels but the differencewas not statistically significant (FIG. 12D). IFN-γ and IL-4 productionwas examined in TSLPR KO mice. Splenocytes from WT and TSLPR KO miceexpressing the DO11.10 transgene were cultured in vitro in the presenceof OVA 323-339. After 4 days of culture, KJ1-26⁺CD4⁺ T cells fromDO11.10/TSLPR KO mice consistently produced more IFN-γ than theanalogous T cells from DO11.10/WT mice (FIG. 12E). Changes in the levelof IL-4 production were difficult to evaluate by intracellular stainingbecause it was so low in WT mice (FIG. 12E). IL-4 levels were measuredby RT-PCR (FIG. 3F). It was found that splenocytes from DO11.10/TSLPR KOmice produced significantly lower levels of IL-4 than DO11.10/WT mice.These results suggest that TSLP can indirectly influence cytokineproduction by CD4⁺ T cells by modulating the activity of DC.

Example 13 TSLPR KO Mice Fail to Develop a Lung Inflammatory Response toOVA Antigen

Because of the ability of TSLP to regulate the immune response of CD4⁺ Tcells, the ability of TSLPR KO mice to control an inflammatory responsewas examined, using the OVA-induced allergic inflammatory response inthe lung (Keane-Myers et al., J Immunol 161:919-926, 1998). WT and TSLPRKO (F4 Balb/c) mice were immunized twice intraperitoneally i.p.) withOVA and challenged by intratracheal (i.t.) and intranasal (i.n.)administration of OVA. As expected in this model, WT mice receiving OVAhad perivascular inflammation and showed marked peribronchiole cuffingwith inflammatory cells, as evidenced by inflammatory cells formingrings around both the vasculature and bronchioles (FIG. 13B versus FIG.13A). PAS staining of the mucoid components in goblet cells revealedthat WT mice also exhibited goblet cell hyperplasia. These mice scored3-3.5 out of 4 on the inflammation scale and showed infiltration ofeosinophils and neutrophils (Table 3) as compared with control WTanimals. In sharp contrast, TSLPR KO mice treated with OVA hadprofoundly fewer cellular infiltrates in the lungs and little to nogoblet cell hyperplasia in the bronchi after antigen challenge (FIG. 13Dversus 13C) and very few inflammatory cells (Table 3). There were noobvious differences between control WT and TSLPR KO mice treated withPBS (FIG. 13C versus 13A, upper panels and FIG. 17 (which is Table 3).

Corresponding to the TSLPR KO mice having a defective “allergic”response, WT and TSLPR KO mice had significantly lower levels of IgE andhigher IgG2a than did WT mice (FIG. 14A). Levels of OVA-specific IgG1,IgG2b, and IgG3 were normal (FIG. 14A). Furthermore, unlike WT mice,TSLPR KO mice sensitized to OVA and subsequently challenged with OVA didnot show a statistically significant increase in IL-2, IL-4, IL-5,IL-10, IL-13, and IFN-γ mRNA as compared to PBS-treated mice (FIG. 14B).These findings are consistent with the greatly diminished infiltrationof inflammatory cells in TSLPR KO animals. Interestingly, OVA induced anincrease in IL-13 mRNA, as evaluated by RT-PCR, in the lungs of TSLPR KOmice, but only to levels seen in PBS-treated WT controls (FIG. 14B).They also displayed a significant increase in IL-12 mRNA (FIG. 14B).These results indicate that TSLP is essential for mounting an optimalpulmonary inflammatory response to OVA.

The greatly diminished inflammatory response in the lungs of TSLPR KOmice may have resulted from the weak activation of CD4⁺ T cells observedor could be due to a developmental defect in the TSLPR KO lungs. To helpclarify the mechanism, WT mice were immunized twice with OVA and totalCD4⁺ T cells were extracted from spleen and LN and transferred (i.v.)into similarly immunized TSLPR KO mice. These mice were challenged byintratracheal (i.t.) and intranasal (i.n.) administration with OVA.Similarly, CD4⁺ T cells from WT and TSLPR KO were transferred into WTand TSLPR KO animals to act as positive and negative controls,respectively. An examination of the lungs revealed severe inflammationand infiltration of eosinophils and neutrophils in WT animals receivingWT CD4⁺ T cells (scoring 3.5 out of 4) (FIG. 15A), while TSLPR KOsupplemented with TSLPR KO CD4⁺ T cells showed little to no inflammation(0-0.5 out of 4) (FIG. 151B), consistent with the results above in Table3 and FIG. 13. TSLPR KO mice that received WT CD4⁺ T cells developedgoblet cell hyperplasia and peribronchiole cuffing, mostly in the largeairways but with some in the medium airways as well. They also showedinfiltration of eosinophils and neutrophils scoring 2.2 out of 4 (range1.5 to 2.75) on the inflammation scale. Thus, the TSLPR KO host can atleast partially support an inflammatory response if WT CD4⁺ T cells wereprovided. This was accompanied by a significant increase in the levelsof IFN-γ, IL-4, IL-5, and IL-13 mRNA in the TSLPR KO mice that receivedWT CD4⁺ T cells as compared to those that received TSLPR KO CD4⁺ T cells(FIG. 16). These results further confirm the critical role that TSLPplays in the activation of CD4⁺ T cells and is consistent with a rolefor this cytokine in the development of the inflammatory response.

The work presented herein demonstrates that TSLP preferentially enhancesthe proliferation and expansion of TCR-stimulated naïve phenotype CD4⁺ Tcells. Moreover, as shown with DO11.10 transgenic mice, TSLP is criticalfor the proper activation of CD4⁺ T cells (e.g. expression of CD69) andtheir transition from naive/resting to activated effector cells.

Memory phenotype CD4⁺ T cells express TSLPR mRNA but showed nosignificant proliferative response to TSLP. The decrease in theresponsiveness of splenocytes from immunized TSLPR KO mice, as comparedto WT animals, to secondary exposure to antigen may be attributed to theweak initiation phase of the immunization process, with fewer cellsdifferentiating into effector and memory cells. It appears that naivecells are poised to respond to TSLP activation.

Murine DC can promote the clonal-specific expansion of naive CD4⁺ Tcells. Although murine DC express TSLPR, this effect of DC on CD4⁺ Tcells was only slightly reduced in the absence of TSLP signaling. Incontrast, the action of TSLP on CD4⁺ T cells was much more critical.Although TSLP had no direct effect on the levels of IFN-γ and IL-4produced by CD4⁺ T cells under neutral or polarizing conditions,indirectly however, TSLP-treated DC diminished the production of IFN-γby CD4⁺ T cells, and correspondingly, more IFN-γ and less IL-4 wereproduced by DO11.10/TSLPRKO splenocytes than by DO11.10/WT cells. Thus,the greatest effect of TSLP on DC may be to augment the differentiationand cytokine production (rather than directly affecting theproliferation of naive CD4⁺ T cells).

The critical role that TSLP plays in mounting an inflammatory responsewas revealed by the diminished response of TSLPR KO mice in anantigen-induced lung inflammation model. Specifically, TSLPR KO micefailed to develop an inflammatory response when challenged with OVA, andlevels of cytokines known to be associated with an allergic immuneresponse (IL-4, IL-5, and IL-10) were not increased. TSLP was slightlyelevated in the lungs of WT animals sensitized with OVA, but thisdifference did not achieve statistical significance Interestingly, thelungs of TSLPR KO mice produced very little IL-13 at rest, althoughlevels increased in response to OVA but only to levels normally observedin un-sensitized WT animals. Serum immunoglobulin levels in theseanimals indicate that a limited immune response is active in TSLPR KOmice since OVA-specific IgG1 and IgG2b were present; however, there wasan increase in IgG2a and a decrease in IgE, corresponding to the morepredominant Th1 phenotype in the absence of TSL. This could potentiallyresult from higher levels of IFN-γ produced in the TSLPR KO CD4⁺ Tcells. Since IL-13 is a mediator of asthma that affects eosinophilicinfiltration, mucus secretion, and airway hyper-responsiveness in thelungs (for example, see Wills-Karp et al., Science 282:2258-2261, 1998),the reduced IL-13 in the lungs of TSLPR KO mice might explain theabsence of inflammation and IgE in these animals. Similarly, the absenceof inflammatory CD4⁺ T cells from the lungs could explain the lack ofIL-5 and subsequently the absence of eosinophils in affected tissues.

The transfer of WT CD4⁺ T cells to TSLPR KO mice had a profound effecton the ability of these mice to mount an inflammatory response. TSLPR KOlungs showed goblet cell hyperplasia and peribronchiole cuffingaccompanied by the infiltration of inflammatory cells, consistent withthe increase in IFN-γ, IL-4, IL-5 and IL-13. Thus, the lungs of TSLPR KOare structurally normal and the micro-environment within them can allowthe development of an inflammatory response when functional preactivatedCD4⁺ T cells are available. However, the inflammation in TSLPR KO micethat received WT CD4⁺ T cells did not reach as severe a level as isobserved in WT animals. This might be explained by the fact that WT micepossess more CD4⁺ T cells, as only a fraction of the WT CD4⁺ T cellsthat were transferred to TSLPR KO animals are expected to be OVAresponsive. In addition, the observed high levels of IL-12 in the lungsof TSLPR KO animals activated with OVA could further contribute tolimiting the extent of the inflammatory response in these animals.

In conclusion, the studies disclosed herein reveal that TSLP plays a keyrole in promoting the proliferation of the naïve population of CD4⁺ Tcells. Although DC activation by TSLP may contribute to this process,the effect of TSLP on CD4⁺ T cells appears to be the more important siteof action. TSLP had little if any direct effect on cytokine productionby CD4⁺ T cells but can influence IFN-γ production via actions on DC.The role of TSLP in an in vivo allergic inflammatory model wasinvestigated. Unexpectedly, profoundly reduced inflammation in the lungsof TSLPR KO mice was found. These data indicate that TSLP signaling iscrucial for generation of an inflammatory reaction in the lung andestablish TSLP as a potential target for modulating inflammation.

Example 14 Exemplary Embodiments

The experiments disclosed above document that TSLP modulates theactivity of dendritic cells and naïve T cells. In addition, it isdemonstrated herein that TSLP diminishes the generation a memory cells.Thus, methods are provided for altering the activity of dendritic cells,naïve T cells, and for diminishing the generation of memory cells, byadministering TSLP, or an agonist thereof, to a subject. Alternatively,a nucleic acid encoding TSLP, or an agonist thereof, can beadministered. Thus, a method is provided for enhancing and/or inducingan immune response in a subject, by administering a therapeuticallyeffective amount of TSLP, or a nucleic acid encoding TSLP to thesubject.

In one example, a method is provided for inducing proliferation of CD4+T cells. The method includes contacting isolated CD4+ T cells with aneffective amount of a TSLP polypeptide or a therapeutically effectiveamount of nucleic acid encoding the TSLP polypeptide, thereby inducingproliferation of the T cells. The T cells can be from any mammal,including, but not limited to a human. The method can include contactingisolated mammalian CD4+ T cells with an effective amount of the TSLPpolypeptide, or a nucleic acid encoding the TSLP polypeptide. SeveralTSLP polypeptides of use are disclosed above. These TSLP polypeptidesinclude an amino acid sequence set forth as one of SEQ ID NOs: 1-5,amino acids 29 to 159 of SEQ ID NO: 1, or amino acids 35 to 159 of SEQID NO: 1.

In another example, a method is provided for treating a subject with animmunodeficiency, such as an immunodeficiency that is the result of aninfection with an immunodeficiency virus (for example, HIV) or that isthe result of a genetic disorder (such as XSCID). The subject can alsoacquire the immunodeficiency as a result of an environmental exposure ofthe administration of an agent, such as radiation or chemotherapy. Themethod includes contacting isolated CD4+ T cells (either autologous orheterologous CD4+ cells) with a therapeutically effective amount of aTSLP polypeptide and administering the CD4+ T cells contacted with theTSLP polypeptide to the subject. The subject can be any mammaliansubject, such as a human subject. Thus, in one example, a method isprovided for treating a subject with an immunodeficiency, byadministering to the subject a therapeutically effective amount of aTSLP polypeptide, or a therapeutically effective amount of nucleic acidencoding the TSLP polypeptide, thereby treating the subject.

In a further example, a method is provided for enhancing an immuneresponse in a subject. The method includes (a) contacting the populationof isolated CD4+ cells (either autologous or heterologous) with acomposition comprising TSLP ex vivo, thereby expanding CD4+ T cells; and(b) introducing the CD4+ T cells into the subject. The method caninclude contacting the population of cells with at least one compositioncomprising an antigen, but does not necessarily include this step. Theantigen can be a viral antigen, a bacterial antigen, or an antigen froma parasite. It should be noted that the subject can be any subject,including but not limited to a human subject. For example, the subjectcan be infected with an immunodeficiency virus, such as HIV-1 or HIV2,or can have an immunodeficiency that is a result of a genetic disorder,such as SCID. The subject can also have acquired an immunodeficiency asa result of an environmental exposure or administration of an agent,such as radiation or a chemotherapeutic agent.

In yet another example, a method is provided for treating a subject withan inflammatory disorder, such as IgE-mediated disorder. The methodincludes administering to the subject a therapeutically effective amountof a thymic stromal derived lymphopoietin (TSLP) antagonist. In oneexample, the IgE-mediated disorder is asthma. In other examples, thedisorder is allergic rhinitis, allergic dermatitis, or allergicconjunctivitis. The TSLP antagonist can be an antibody that binds TSLPor the TSLP receptor, such as a humanized antibody, and can beadministered by any means, such as by inhalation, intranasal, and/orintratrachael administration. In addition to the TSLP antagonist thesubject can be treated with therapeutically effective amount of ananti-infective agent, an anti-inflammatory agent, a bronchodilator, anenzyme, an expectorant, a leukotriene antagonist, a leukotrieneformation inhibitor, or a mast cell stabilizer.

Example 15 Rhino-Conjunctivitis Model System

Adult mice (6-8 weeks in age, of either sex) are sensitized byintraperitoneal injection of 50 micrograms of ragweed mixed 1:1 withaluminum hydroxide on days 0 and 7. Mice are challenged by eye drops andintranasally with ragweed in PBS 5 microliters (1 mg) in the eye and 30microliters (50 μg) in the nose on days 14 and 15. Animals aresacrificed on day 17.

One group of animals are treated with a TSLP antagonist 24 hours priorto antigen challenge (day 13). For example, mice are treated 24 hoursprior to challenge with an antibody that binds TSLP. An exemplary amountis 1 mg i.p. Control animals are treated with isotype control antibody,and a group is treated with PBS.

Mice are then assessed for an inflammatory response. For example, eyeswith lids attached are isolated from the animals. The nasal passages arealso removed. Histology is performed, such as periodic Schiff's staining(PAS) to identify goblet cells. Hemotoxylin and eosin and Wright'sGiemsa stain are can also be used. Mast cell degranulation, eosinophilinfiltration and goblet cell hyperplasia are assessed. Mice administeredthe TLSP antagonist show reduced mast cell degranulation and/or reducedeosinophil infiltration and/or reduced goblet cell hyperplasia.

It will be apparent that the precise details of the methods orcompositions described may be varied or modified without departing fromthe spirit of the described invention. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow.

1. A method for inducing proliferation of CD4+ T cells, comprising contacting isolated CD4+ T cells with an effective amount of a thymic stromal derived lymphopoietin (TSLP) polypeptide or a therapeutically effective amount of nucleic acid encoding the TSLP polypeptide, thereby inducing proliferation of the T cells.
 2. The method of claim 1, wherein the method comprises contacting isolated mammalian CD4+ T cells with an effective amount of the TSLP polypeptide.
 3. The method of claim 1, wherein the TSLP polypeptide comprises an amino acid sequence set forth as one of SEQ ID NOs: 1-5, amino acids 29 to 159 of SEQ ID NO: 1, or amino acids 35 to 159 of SEQ ID NO:
 1. 4. A method of inducing or enhancing an immune response in a subject, comprising contacting isolated CD4+ T cells with a therapeutically effective amount of a TSLP polypeptide; and administering the CD4+ T cells contacted with the TSLP polypeptide to the subject thereby treating the subject.
 5. The method of claim 4, wherein the subject has an immunodeficiency.
 6. The method of claim 4, wherein the TSLP polypeptide comprises a polypeptide set forth as one of SEQ ID NOs: 1-5, amino acids 29 to 159 of SEQ ID NO: 1, or amino acids 35 to 159 of SEQ ID NO:
 1. 7. The method of claim 5, wherein the immunodeficiency is the result of an infection with an immunodeficiency virus.
 8. The method of claim 7, wherein the subject is human, and wherein the immunodeficiency virus is a human immunodeficiency virus (HIV).
 9. The method of claim 5, the subject has an immunodeficiency as a result of a genetic disorder.
 10. The method of claim 5, wherein the immunodeficiency is a result of treatment with radiation, a chemotherapeutic agent, or a combination thereof.
 11. The method of claim 4, comprising contacting the CD4+ cells with at least one composition comprising an antigen.
 12. The method of claim 11, wherein the antigen comprises a viral antigen, a bacterial antigen, or an antigen from a parasite.
 13. A method of treating a subject with an immunodeficiency, comprising administering to the subject with the immunodeficiency a therapeutically effective amount of thymic stromal derived lymphopoietin (TSLP) polypeptide, or a therapeutically effective amount of nucleic acid encoding the TSLP polypeptide, thereby treating the subject.
 14. The method of claim 13, wherein the subject is infected with an immunodeficiency virus.
 15. The method of claim 14, wherein the subject is a human and where the immunodeficiency virus is a human immunodeficiency virus (HIV).
 16. The method of claim 13, wherein the subject has an immunodeficiency as a result of a genetic disorder.
 17. The method of claim 13, wherein the immunodeficiency is a result of treatment with radiation, a chemotherapeutic agent, or a combination thereof.
 18. A method of treating a subject with an IgE-mediated disorder, comprising administering to the subject a therapeutically effective amount of a thymic stromal derived lymphopoietin (TSLP) antagonist, thereby treating the subject.
 19. The method of claim 18, wherein the IgE-mediated disorder is asthma.
 20. The method of claim 18, wherein the TSLP antagonist is an antibody that binds TSLP or the TSLP receptor.
 21. The method of claim 20, wherein the antibody is a humanized antibody.
 22. The method of claim 18, further comprising administering a therapeutically effective amount of an anti-infective agent, an anti-inflammatory agent, a bronchodilator, an enzyme, an expectorant, a leukotriene antagonist, a leukotriene formation inhibitor, or a mast cell stabilizer.
 23. The method of claim 18, wherein the TSLP antagonist is administered by inhalation.
 24. The method of claim 18, wherein the disorder is rhino-conjunctivitis or allergic dermatitis. 